List of proteins utilized on the analysis of sequence alignment
\r\n\tIn sum, the book presents a reflective analysis of the pedagogical hubs for a changing world, considering the most fundamental areas of the current contingencies in education.
",isbn:"978-1-83968-793-8",printIsbn:"978-1-83968-792-1",pdfIsbn:"978-1-83968-794-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"b01f9136149277b7e4cbc1e52bce78ec",bookSignature:"Dr. María Jose Hernandez-Serrano",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10229.jpg",keywords:"Teacher Digital Competences, Flipped Learning, Online Resources Design, Neuroscientific Literacy (Myths), Emotions and Learning, Multisensory Stimulation, Citizen Skills, Violence Prevention, Moral Development, Universal Design for Learning, Sensitizing on Diversity, Supportive Strategies",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 14th 2020",dateEndSecondStepPublish:"October 12th 2020",dateEndThirdStepPublish:"December 11th 2020",dateEndFourthStepPublish:"March 1st 2021",dateEndFifthStepPublish:"April 30th 2021",remainingDaysToSecondStep:"3 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Phil. Maria Jose Hernandez Serrano is a tenured lecturer in the Department of Theory and History of Education at the University of Salamanca, where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. in Education and Training in Virtual Environments by research with the University of Manchester, UK (2009).",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"187893",title:"Dr.",name:"María Jose",middleName:null,surname:"Hernandez-Serrano",slug:"maria-jose-hernandez-serrano",fullName:"María Jose Hernandez-Serrano",profilePictureURL:"https://mts.intechopen.com/storage/users/187893/images/system/187893.jpg",biography:"DPhil Maria Jose Hernandez Serrano is a tenured Lecturer in the Department of Theory and History of Education at the University of Salamanca (Spain), where she currently teaches on Teacher Education. She graduated in Social Education (2000) and Psycho-Pedagogy (2003) at the University of Salamanca. Then, she obtained her European Ph.D. on Education and Training in Virtual Environments by research with the University of Manchester, UK (2009). She obtained a Visiting Scholar Postdoctoral Grant (of the British Academy, UK) at the Oxford Internet Institute of the University of Oxford (2011) and was granted with a postdoctoral research (in 2021) at London Birbeck University.\n \nShe is author of more than 20 research papers, and more than 35 book chapters (H Index 10). She is interested in the study of the educational process and the analysis of cognitive and affective processes in the context of neuroeducation and neurotechnologies, along with the study of social contingencies affecting the educational institutions and requiring new skills for educators.\n\nHer publications are mainly of the educational process mediated by technologies and digital competences. Currently, her new research interests are: the transdisciplinary application of the brain-based research to the educational context and virtual environments, and the neuropedagogical implications of the technologies on the development of the brain in younger students. Also, she is interested in the promotion of creative and critical uses of digital technologies, the emerging uses of social media and transmedia, and the informal learning through technologies.\n\nShe is a member of several research Networks and Scientific Committees in international journals on Educational Technologies and Educommunication, and collaborates as a reviewer in several prestigious journals (see public profile in Publons).\n\nUntil March 2010 she was in charge of the Adult University of Salamanca, by coordinating teaching activities of more than a thousand adult students. She currently is, since 2014, the Secretary of the Department of Theory and History of Education. Since 2015 she collaborates with the Council Educational Program by training teachers and families in the translation of advances from educational neuroscience.",institutionString:"University of Salamanca",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Salamanca",institutionURL:null,country:{name:"Spain"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"23",title:"Social Sciences",slug:"social-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"301331",firstName:"Mia",lastName:"Vulovic",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/301331/images/8498_n.jpg",email:"mia.v@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6942",title:"Global Social Work",subtitle:"Cutting Edge Issues and Critical Reflections",isOpenForSubmission:!1,hash:"222c8a66edfc7a4a6537af7565bcb3de",slug:"global-social-work-cutting-edge-issues-and-critical-reflections",bookSignature:"Bala Raju Nikku",coverURL:"https://cdn.intechopen.com/books/images_new/6942.jpg",editedByType:"Edited by",editors:[{id:"263576",title:"Dr.",name:"Bala",surname:"Nikku",slug:"bala-nikku",fullName:"Bala Nikku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"52789",title:"Programmed Cell Death-Related Proteases in Plants",doi:"10.5772/65938",slug:"programmed-cell-death-related-proteases-in-plants",body:'\nEvery proteinaceous enzyme that performs hydrolysis of peptide bonds is correctly designed as protease (peptidase) [1, 2]. This term was first used by Vines [3] based on direct and indirect evidences from studies with algae, some fungi and Phanerogams. Long before this work, the word “proteolytic” was applied by Roberts [4] to describe the digestive process on human stomach, and in the first years of the twentieth century, the digestive proteins trypsin and pepsin as well as other autocatalytic enzymes were known as proteases, being pepsin credited to do “proteolysis” since 1877 [5]. In 1928, Grassmann and Dyckerhoff [6] established important definitions concerning the nature of the catalytic activities of proteases. In resume, the peptide cleavage pattern by proteases can be internal, for endopeptidases; on the N‐terminal portion, for aminopeptidases; or C‐terminal, for carboxypeptidases [2].
\nDespite this difference, all proteases share the same catalytic strategy, as they polarize the carbonyl group on the peptide bond of the substrate by the stabilization of the oxygen atom on an oxyanionic hole, what makes the carbon atom more vulnerable to the attack of an activated nucleophile. The nature of the nucleophile presents some variation and is determinant to the employed mechanism for enzymatic catalysis. As a result of these variations, the proteases are grouped into six different catalytic families: serine, threonine, cysteine, aspartic, glutamic and metallopeptidases (http://merops.sanger.ac.uk/). The larger families (cysteine, serine, aspartic and metallopeptidases) have a wide range of distribution on living organisms, and are also present in the “controversial” group of viruses [7].
\nAbout their function, these enzymes are well known for the promotion of protein degradation on amino acid unities. Besides this function, they regulate the destination of other proteins based on their cleavage specificity and participate on important cellular pathways, being key regulators in different response processes to environmental factors and developmental signals [2].
\nOne of these cellular pathways is the event of programmed cell death (PCD) that, since the 2000s, is being studied with increased interest on non‐metazoan organisms, in particular on plant models. On metazoan, one of the main constituent of the pathway are cysteine proteases known as caspases. They are not found in plants, but an increasing number of reports shows strong evidences that caspase-related proteases that belong to the metacaspase group, as well as proteases with caspase-like activities are eminent on plant PCD [8].
Programmed cell death (PCD) is a genetically controlled physiological innate mechanism, which involves the selective death of individual cells, tissues or entire organs. It is a process different from necrosis as it occurs passively in response to environmental perturbations [9]. Together with the chromatin remodelling machinery, the cell cycle regulation mechanisms, the nuclear envelope and the cytoskeleton, this process is one of the major eukaryotic innovative aspects, which allowed the development of more complex organisms [10].
\nMolecular evidences have pointed out that the PCD machinery has evolved since very early stages of the evolutive history, and that this evolution has been processed through expansion and innovation of protein recruitment domains, as well as through the derivation of effector domains and horizontal gene transference events [11].
\nThe best‐understood models in PCD are metazoan organisms and in concern to the cell morphology and involved organelles, two main categories are known: apoptosis and autophagy [12–14].
\nApoptosis is defined by three main morphological characteristics: nuclear DNA fragmentation, apoptotic bodies’ formation and degradation of the apoptotic bodies on the lysosome of a phagocytic cell [12–14].
\nAutophagy is the main system of degradation and recycling in eukaryotic cells, contributing to the clearing of cellular compounds and cytosolic portions. This process can occur in two forms: through the cytosolic sequestration by the vacuole or through the sequestration of large portions of cytosol by a structure called autophagosome [15–17].
\nOn situations where a cellular set is under a more intense stress, so that the cells are not able to activate the apoptotic PCD pathway, cell death occurs through necrosis, characterized by a protoplasmic swelling due to the loss of the osmoregulation control capability, and consequent water and ions migration to the cell [18].
\nIn plants, PCD is observed under diverse circumstances through the entire life cycle of many species, as well as in response to biotic and abiotic stimuli, what allows wide biochemical and developmental plasticity [19], as, for example:\n
on the degeneration of cells from tissues with transitory functions, such as cotyledons, suspensor, certain leaves, petals [20] and secretory tissues [21];
on the elimination of excessive produced cells, as in the case of some unisexual flowers that initially produce male and female organs, and must eliminate one of these groups to become functional [22].
Concerning to PCD, the main group of proteases performing important roles is the subfamily C14 from the CD clan of cysteine proteases. Their representatives include the metacaspases, paracaspases and caspases.
\nThe first discovered and the most known in terms of structure and function are the caspases. The early reports of caspases and related proteins are those with the genes cd‐3 and cd‐4, as well as their encoded products from Caenorhabditis elegans and the enzyme caspase‐1 from mammals [23–27].
\nTo date, caspases have been proven to be “in the heart” of a pathway that mediates the highly ordered process of apoptotic cell suicide [28], and, indeed, they are the convergence point of biochemical pathways on cellular substrates which lead to the activation of a “protease cascade” (the caspase cascade).
\nThe importance of this cascade can be seen when disturbance of its regulation occurs on the cells, what causes immunodeficiency, carcinogenesis and other troubles related to aberrant PCD [29–31].
\nThe cascade overview reveals two functional groups of caspases, concerning their position on the sequence of proteolytic events (Figure 1). The first caspases to be activated are the initiators which become active by the “induced proximity mechanism”, often triggered by recruitment of adaptor protein complexes, such as recruitment domain of membrane receptors or by a huge protein complex called apoptosome. This induced proximity leads to the oligomerization of caspase molecules which form a heterodimer that becomes able to trigger the activation of the effector caspases by cleavage of a pro‐domain, since these enzymes are synthetized as zymogens. After this cleavage, the effectors themselves also stay organized as heterodimer, and cleave the apoptotic substrates, leading to a typical cellular morphology [8].
Representation of caspase, metacaspase and paracaspase major domains. IC: initiator caspase; EC: executor caspase; MI: type I metacaspase; MII: type II metacaspase; PC: paracaspase. Red: p20 domain; blue: p10 domain; orange: prodomain/recruitment domain; purple: death domain; green: immunoglobulin‐like domain; yellow: paracaspase C‑terminal region. Based on Vercammen et al. [8].
Despite the positional and functional differences, all caspases belong to a group named ICE family (interleukin‐1β‐converting enzyme/caspase 1 family), since caspase 1 was the first characterized member.
\nAbout the phylogeny, these molecules are unique and well distributed among the Animalia Kingdom. Their presence ranges from vertebrate organisms such as Homo sapiens and Danio rerio to invertebrates such C. elegans and Drosophila melanogaster [32]. Also, their involvement with cell death processes is also well documented.
\nAlongside caspases, on clan CD, there are caspase relatives called paracaspases which are found in metazoans and in slime molds [4, 32] (Figure 1). In humans, paracaspase/MALT1 is associated to the lymphocyte activation by the NF‐kB pathway [33, 34]. In Dictyostelium, the paracaspase gene disruption does not affect the developmental cell death process, what leads to the suggestion that the enzyme is not required for the phenomenon [35]. A function for human paracaspase MALT 1 on caspase‐8 modulation is reinforced by Kawadler et al. [36], whose data show that MALT1 activates caspase‐8 during TCR signalling but reduces the cleavage of caspase‐3, avoiding apoptosis and inducing cell proliferation.
\nAbout its structural architecture, the human MALT 1 monomer has an apparent molecular mass of 41 kDa and the dimer, about 84 kDa and the overall predicted structure shows an N‐terminal death domain, two immunoglobulin domains, a paracaspase domain and another immunoglobulin‐like domain. The paracaspase domain is folded in a similar way to that of caspases and exhibits the ability to bind on substrate. Also, the enzyme, contrary to caspases, seems not to require cleavage of loop 4 to become active [37, 38].
\nThe other member of CD clan of the cysteine proteases are the metacaspases (Figure 1). They are found in Fungi, Protozoa, Chromista and Plantae [8], and were first described in 2000 [32], on a study performing structural and sequence analysis, which revealed a great diversity of protease genes related to caspases in these Phyla. They were credited as strong candidates to perform central roles on PCD [11].
\nBased on their structural architecture, they were divided into two categories: type I and type II metacaspases. The type I subclass metacaspases have an N‐terminal extension, a prodomain with a proline‐rich repeat motif that is absent on the type II metacaspases, which, instead, present a 200 amino acid C‐terminal extension. Also, many plant type I metacaspases have a zinc finger motif that is similar to that of the plant hypersensitive response/cell death protein lsd‐1 [39].
\nRecently, a third group, named as type III metacaspases, has been proposed [40]. These proteins are found on many phytoplankton organisms and are not grouped with the other metacaspase groups, probably by a p10 domain rearrangement on which the motif SGCXDXQTSADV is located on the N‐terminus rather than on the C‐terminus, as usual for plant metacaspases. The studied organisms also revealed the presence of metacaspase‐like proteases which possess only the p10 domain. These proteases are found on bacteria and may represent an evolutionary connection.
After the first finding of caspase‐like activities on plants [41], as well as on other non‐metazoan organisms, an extensive search for the enzymes responsible for these activities was performed [32]. For the model plant, Arabidopsis thaliana, for example, nine metacaspases encoded by its genome were found: 3, of type I and 6, of type II metacaspases [42]. The list is being extended, among other examples are: soybean (Glycine max) with 16 metacaspases (10, type I and 6, type II); wheat (Zea mays) with 12 metacaspases (8, type I and 4, type II) and Oryza sativa sp japonica with eight metacaspases (4, type I and 4, type II) [43].
\nThe first observations using cell extracts suggested that metacaspases could be responsible for the caspase‐like activities found in plants [44]. Nevertheless, recorded reports show that plant metacaspases are unable to cleave caspase synthetic substrates [45–47]. The caspases cleave their substrates after aspartate residues and metacaspases cleave after Asp or Lys residues at P1 position [43]. So, appears that the metacaspases are not directly responsible for the caspase‐like activities found in plants.
\nAlthough metacaspases do not have caspase‐like activities, many works suggest that they have a role in PCD [48]. The inhibition of a type II metacaspase (McIIPa) suppressed PCD in suspensor cells from an embryonic culture from Picea abies. During P. abies embryogenesis, it was shown that McIIPa is transported from cytosol to nuclei, where its presence was correlated to DNA fragmentation detection. Also, through experiments with a cell free system in the presence of a mutant form of this protein on its active site, the nuclear alterations were not detected [44]. These data reinforce that McIIPa is directly involved on a pathway which generates nuclear degradation, an event present on most eukaryotic PCD programmes. In this pathway, the protease can play a role on the cleavage of nuclear proteins, similar to what happens to the metazoan effector caspases [46].
\nThe heterologous expression of the A. thaliana metacaspases MCP1b and MCP2b on a Saccharomyces cerevisiae strain, disrupted for its metacaspase YCA1 gene, was able to trigger an apoptosis‐like phenotype under hydrogen peroxide treatment, and this effect was seen to be abolished on the presence of the pancaspase inhibitor based on the sequence VAD. These findings reinforce a metacaspase role on PCD, as well as the requirement of proteins with caspase‐like activity [42].
\nThe involvement of metacaspases on PCD is also suggested by works with plants under pathogen attack. Some examples are given by the detection of gene expression for MCA1 on A. thaliana leaves infected by Pseudomonas syringae [49], and by the increasing levels of a type II metacaspase on tomato leaves under Botrytis cynerea infection, with the detection of cell death phenotype [50].
\nIt cannot be excluded, though, that metacaspases could not be directly involved on PCD regulation, but indirectly involved on signalling cascades that leads to PCD [48].
\nIn face of this, the role of metacaspases are still under discussion, as well as their classification, since there are evidences which favour, and others contrary, to their groupment together with caspases [51, 52]. A cascade mechanism comparable to that of caspases was proposed for vegetable systems concerning cysteine proteases on senescent leaves and seeds on maturation. Bozhkov et al. [46] reinforced the idea that the execution of PCD in plants is controlled by two groups of enzymes with separated cellular localization. One of them is accumulated on lytic compartments and vacuoles, and the other has cytoplasmic‐nuclear localization, as in the case of MCIIPa.
The first report of a metacaspase on yeast was made by Madeo et al. [53], where the overexpression of the protein codified by theYor197w gene stimulated PCD associated‐caspase‐like activity on S. cerevisiae cells, under H2O2 stress. The authors proposed the name Yeast Caspase‐1 (YCA1) for the protein. Also, a S. cerevisiae mutant strain with an inactive gene encoding a deubiquitinating enzyme (UBP10) showed an apoptosis‐related phenotype which appears to be related to the YCA1 presence, in a manner similar to that observed when the cells were submitted to external stimuli, such as treatment with H2O2. The overexpression of YCA1 in cells lacking UBP10 resulted on a decrease of about 53% in viability [54].
\nA study with frataxin‐deficient yeast cells (Δyfh 1), sensible to pro‐oxidant chemicals, showed that YCA1 is induced under H2O2 stress. When performance of metacaspase‐deficient yeasts (Δyca1) was evaluated under oxidative stressing condition, a resistance to cell death, reflected by higher glutathione concentrations than the wild‐type, was detected, probably caused by the absence of the metacaspase [55].
\nBy using an original approach of combining the techniques of a digestome analysis (an in vitro assay aimed to search putative specific substrates of proteases), cleavage of recombinant GAPDH by metacaspase and evaluation of protein levels in vivo, wild‐type and YCA1 overexpressing yeast cells upon H2O2‐induced apoptosis were evaluated. Under these oxidative conditions, the enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), was shown, by mass spectrometric analysis, to be a specific target of metacaspase. This result suggests a link between programmed cell death and metabolism [56]. Besides the potential role in MCP, this metacaspase seems to have involvement on cell cycle regulation. Deficient yca1 yeast revealed an increased level of proteins related to the vacuolar catabolism, including six peptidases, as well as chaperones involved on stress response and protein aggregates elimination. YCA1 was also copurified with chaperones that respond to protein aggregation and with a cell‐cycle control protein; thus, a role on clearance of protein aggregates during mitosis and aging was proposed [57]. This potential on protein clearance is dependent on the presence of Hsp40 and Hsp104 chaperones, and is reduced by deletion of the proteasome regulator Rnp4, which reduces the proteasome levels. When overproduced, YCA1 extended the life span of the cells by 46–56% [58].
\nMetacaspase studies are also being performed with other fungi, as in the case of Candida albicans metacaspase 1 (CaMCA1). Mutant strains for this gene showed a lack of PCD under oxidative stress, as well as a decrease of energy metabolism intensity followed by accumulation of trehalose and increase on expression of genes related to trehalose biosynthesis, what is a well‐known protective mechanism against oxidative stresses [59]. Other example is the study of two metacaspases from Aspergillus fumigates, whose deficiency was associated with loss of membrane phospholipid asymmetry and an increased growth under conditions of endoplasmic reticulum stress [60].
Among the first reports concerning metacaspases on protists was the work of Szallies et al. [61]. According to this, Trypanosoma brucei encodes five putative metacaspases. The expression of the T. brucei metacaspase MCA4 in S. cerevisiae caused a PCD‐related phenotype, and the protein was found to be located on nuclei, alongside the yeast metacaspase MCA1. When overexpressed, MCA4 induced PCD.
\nTwo metacaspases (LdMC1 and LdMC2) of Leishmania donovani were also characterized and displayed high homology among each other. The proteins were seen to be capable of cleaving trypsin substrates and were inhibited by classical trypsin inhibitors such as leupeptin and antipain. Despite not being able to cleave caspase substrates, when their genes were overexpressed in the parasite cells, a higher sensibility to PCD was observed [62]. The metacaspases MCA2, MCA3 and MCA 5 were detected in vivo in their full molecular form and seem not to suffer processing to become active in the parasite cells. They were found to be associated with recycling endosomes that contain recycling receptors and PCD‐independent functions were proposed for this association with endosomes [63].
\nThe capability to induce PCD was also investigated for the Leishmania major single metacaspase (LmjMCA), whose heterologous expression in a S. cerevisiae defective strain was successful on the induction of PCD by oxidative stress. The protease originated from the metacaspase gene revealed an arginine‐specific activity and through approaches of site‐directed mutagenesis, it was possible to determine that its activity is dependent of H147 and C202 [64]. This metacaspase was suggested to be essential for the parasite, since the tentative to obtain a null mutant was unsuccessful. The protein was detected in variable levels in different compartments at different stages of the cell cycle, in association with the dividing kinetoplast and the nuclear spindle, what may suggests a role on DNA segregation. Also, it was seen that the protein was associated with cytoskeleton filaments. A role on differentiation and proliferation has been additionally proposed [65].
\nThe activity of a Plasmodium falciparum metacaspase 1 (PfMCA‐1) increased under high concentrations of calcium and induced PCD under stress conditions [66]. The authors have also presented evidences of the ability of PfMCA1 of triggering a downstream enzyme that is sensible to the pancaspase inhibitor z‐VAD‐fmk.
Caspases, metacaspases and paracaspases have a conserved pattern of tridimensional organization and are then considered as structural homologues. The degree of this conservation is variable, but the overall structure is related to a conformation named caspase fold that is characterized by a core formed by a contiguous six‐stranded β‐sheet (β1–β4, β7 and β8) and helices α1–α5 region, present in every caspase structure. Also, the presence of three well‐ordered loops (L1, L2 and L4) is well characterized [67].
\nAs it was early discussed, metacaspases are divided into two groups based on the presence of a pro domain or a linker region. Type I metacaspases possess an N‐terminal prodomain with length of about 80–120 amino acids [32], with two CXXC‐type LSD1‐like zinc finger structures as well as a proline/glutamine rich region [45, 68]. The type II metacaspases do not have prodomains but, otherwise, contain a large loop (linker region) between the p10 and p20 domains which ranges about 90–150 amino acids [32, 69].
\nThe p10 and p20 domains are present in all of these proteins. For p10, there is a SGCXDXQTSADV consensus sequence, as well as other conserved short sequences [40]. The p20 domain contains the conserved catalytic dyad histidine/cysteine as a remarkable feature, where the two amino acids are distant from each other in about 29–47 amino acids. There are also other conserved regions/amino acids that together give about 80% of consensus on the entire sequence [32]. A noticeable signature is the motif DSCHSG in the surroundings of the catalytic Cys, which is highly conserved among all type II plant metacaspases [70].
\nDifferently of metacaspases, all caspases contain a conserved QACXG (where X can be R, Q, or G) pentapeptide active‐site motif. The catalytic residues histidine 237 and cysteine 285, and those involved in forming the P1 carboxylate binding pocket on caspase 1 (Arg‐179, Gln‐283, Arg‐341 and Ser‐347), are also conserved in all other caspases, except for the conservative substitution of the threonine for the serine 347 in caspase 8. This explains the requirement for an aspartate in the substrate P1 position. The residues that form the P2–P4 binding pocket are not well conserved, suggesting that they may determine the substrate specificities of the different caspases [71]. The metacaspases do not have these features and present cleavage specificity for lysine/arginine on the P1 position on the substrate, so their binding residues seem to be of opposite chemical nature from those of caspases.
\nThe type II metacaspases present autoprocessing sites, whose cleavage seems to be necessary for their full activation: the residues Lys 260 and Arg 214, on the wheat type II metacaspase [70]; Lys 269, from MCIIPa [46]; and the Arg 214, from AtMC9 [45]. Caspases, otherwise, have different cleavage site, always after Asp residues. Caspase 1 is cleaved after Asp‐103, Asp‐119, Asp‐297 and Asp‐316 [71]; caspase 7, after Asp‐23, Asp‐198 [72]; and caspase 3, after Asp‐9/Asp‐28 and Asp‐175 [73].
\nActually, there are only two metacaspases with elucidated structural organization. MCA2, from T. brucei, the first metacaspase with elucidated structure, is topologically more related to other metacaspases, such as YCA1, than to caspases, presenting the same number and organization of β sheets and α helix, although the enzyme showed 65% of secondary elements similarly to caspase 7. Also, the β‐sheet region of MCA2 is sized two strands longer than caspases. As for YCA1, this characteristic prevents dimerization. Autocatalytic processing occurs only in vitro and is not required for enzyme activity, which was also shown to be dependent of calcium as in other metacaspases [74].
\nThe crystal structure from the yeast metacaspase Yca1 shows the general patterns of the caspase fold, with the well‐ordered loops, being L1 and L4 in opposing sides of the substrate interaction site, and the conserved caspase core. Concerning β conformations, yeast YCA1 presents two β‐strands (β5 and β6) which are absent in caspases (caspase 3 and caspase 9), and these are located in a way that blocks dimerization. As a result, YCA1 cannot form dimer, as caspases do. The catalytic dyad consists on the residues Cys276 and His220 which are well conserved among other proteins with caspase fold. Also, the identity between the YCA1 and the caspases 3 and 9 is lower than 12%, and the sequence divergence greatly affected the root‐mean‐square deviation (RMSD) analysis. The superimposition of YCA1 and caspase 3 showed a higher structural variation than the superimposition of caspase 3 and 9. On in vitro assays, the presence of Ca2+, and not of other divalent cations, was required to an enhanced capability of auto processing. Under this condition, two peptide fragments were formed revealing that the processing occurred after Arg72 and Lys86. When compared with MCA2, YCA1 shows 24.9% of amino acid sequence identity and a very similar structure through RMSD analysis [75].
Despite the growing knowledge about the structure and function of CD clan members of cysteine proteases, the molecules involved on the control of their activities are still to be more unravelled. As discussed before, one of the main activation mechanisms of the caspases is its cleavage on specific sites, promoted by other caspases. This processing results on conformational changes and dimerization that enhance the substrate cleavage activity of the target caspase.
\nFor this to happen, it is necessary the assemblage of huge protein complexes, which function as activation platforms. Examples of these are the Fas death‐induced signalling complex (DISC), whose association is required for the caspase 8 (pro‐caspase 8) self‐activation and the apoptosome, which binds to caspase 9 (pro‐caspase 9) prior to its activation [11, 28, 31]. It is not clear, if in the case of metacaspases, the auto processing and dimerization are always required for their activation as well as the formation of protein complexes [8].
\nUntil now, biochemical studies have demonstrated that only type II metacaspases undergo autocatalytic activation, similar to the phenomenon observed for caspases. Contrary to that, no proteolytic activation was observed for type I metacaspases. So a scenery, where type I prometacaspases become active by the action of death signals coupled to the activation of type II prometacaspases, which by their turn become active and able to cleave other proteases and trigger the degradation of cellular components, was proposed as a probable signal transduction pathway during PCD proteolysis in plants [69] (Figure 2).
Proposed cascade model of plant proteolytic events triggered by a death inductor signal. The type II metacaspase inhibitors (red arrow) and stimulators (green arrow) are displayed, as well as possible substrates.
Alongside with the proteolysis processing requirements, there are reports which show calcium dependence for type II metacaspases. By in vitro experiments, a type II recombinant metacaspase from A. thaliana exhibited Ca2+ dependence (10 mM) for its activation, on a pattern indicative of auto‐processing [76]. A similar Ca2+ dependence was detected on in vitro assays with the T. brucei MCA2, whose activity peaks on the presence of 10 mM of Ca2+, although the enzyme does not require autoprocessing [77]. This characteristic was shown to be valid for both L. major and P. falciparum metacaspases [66].
\nOne candidate to be a metacaspase regulator is a serine protease inhibitor called Serpin. Serpin1 from A. thaliana was shown to have a potent cleavage activity towards the reactive centre loop of metacaspase 9, besides the ability of covalent binding to the target protein on in vitro assays. This study has also demonstrated that both proteins were localized on extracellular space, suggesting they could interact under in vivo conditions as well [78].
\nOther control mechanism proposed is the S‐nitrosylation, that is used as a regulation strategy of certain proteins under basal NO levels. The S‐nitrosylation of the catalytic cysteine (Cys‐147) on A. thaliana metacaspase 9 (AtMC9) on its mature processed form does not affect its activity. This happens because other cysteine (Cys‐29) residue can act as alternative nucleophile. Despite this, the enzyme can be kept inactive through S‐nitrosylation, and otherwise, become active only under conditions of disturbance on cellular redox balance [79].
\nThe effect of zinc on metacaspase activity was also investigated. The supplementation of plant embryos with extra zinc suppressed the terminal differentiation and death of the suspensors, delaying the embryo maturing, and also reducing the intensity of metacaspase activity between 96 and 168 h of development, which is the period when the suspensor death occurs [80]. These data, alongside the work of Bozhkov et al. [46], suggest that zinc may be part of a mechanism of posttranslational regulation of metacaspases, still to be further examined.
Little is known about the metacaspase natural substrates, to the present date. This lack of information makes hard to construct a PCD pathway as was done for caspases. Despite this, the efforts aiming at the elucidation of this question are growing.
\nThe first biological substrate discovered for a metacaspase is the Tudor Staphylococcal Nuclease (TSN), a protein involved on gene expression regulation, highly conserved phylogenetically. The cleavage of this protein prevents its function and is important for the execution of apoptosis; also, the protein is known to be a part of the human caspase 3 degradome. TSN was shown to be cleaved by the metacaspase McIIPa (type II metacaspase of P. abies) on an in vitro assay. In vivo, the McIIPa activity was shown to be simultaneous to the decrease of TSN activity, what was deduced from TUNEL positive embryonic cells [81].
\nAnother substrate found for a metacaspase was the already mentioned GAPDH. This protein was detected as a digest product of an YCA1 metacaspase‐enriched extract from S. cerevisiae cells subjected to H2O2 treatment. As recombinant YCA1 was shown to cleave GAPDH in vitro, the in vivo evaluation of the GAPDH performed during a comparison between the wild‐type and a mutant yeast strain, disrupted for YCA1, both under PCD triggering condition, showed a reduction of the enzyme levels on the wild‐type (resistant to PCD). The GAPDH is also a caspase substrate, but the cleavage again happens on different sites of those targeted by the metacaspases. Yet, this is another evidence for the existence of conserved molecular members of a PCD pathway in metazoan and in metacaspase‐bearing organisms [56].
\nRecently, as a remarkable effort, a proteome‐wide‐level study of A. thaliana seedlings, focusing on the identification of physiologic substrates of metacaspase 9, has been performed employing a digestome analysis strategy. Important features of the target proteins were prospected, and it was possible to map the frequencies of the amino acids sitting at the neighbourhood of the Arg or Lys P1‐specific cleavage sites. Along with other interesting features, the enzyme has shown a strong tendency to prefer acid residues, as Asp and Glu at the P1’ position. Among the identified substrates, was phosphoenolpyruvate carboxykinase 1 (PEPCK1), a gluconeogenesis enzyme. This protein was shown to be cleaved in vivo in such a manner that its activity was enhanced, and thus, the glucose de novo synthesis pathway may be stimulated during PCD [82].
For the comprehension of the structural organization of a type II metacaspase, the delimitation of the p20 and p10 domains of the metacaspase 4 from G. max was performed by our group, as well as the analysis of its catalytic amino acids residues and the motifs conservation with other metacaspases and caspases, through protein alignment. Also, the tridimensional structure of the protein was predicted. Metacaspase and caspase sequences of organisms from different taxa (Table 1) were aligned using the software Clustal X [83] (http://www.clustal.org/). The sequences were obtained from the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/) data bank and given a treatment for removal of prodomains and loops, for adjustment to the alignment. In this process, the works of Vercammen et al. [8] and of Uren et al. [32] were used as a guide to the delimitation of the domains and catalytic residues.
Protein | \nSpecie | \nIdentification code (gi) | \n
---|---|---|
Caspase8 | \nHomo sapiens | \n12862693 | \n
Caspase8 | \nMus musculus | \n29436722 | \n
Caspase8 | \nGallus gallus | \n16555407 | \n
Caspase8 | \nXenopus laevis | \n148228484 | \n
Caspase8 | \nDanio rerio | \n46397548 | \n
CED3/NEDD2‐like | \nDrosophila melanogaster | \n220901640 | \n
CED3 | \nCaenorhabdits elegans | \n11967321 | \n
Metacaspase4 | \nGlycine max | \n356556698 | \n
Type II metacaspase | \nNicotiana tabacum | \n195963550 | \n
Metacaspase8 | \nArabidopsis thaliana | \n32482822 | \n
Metacaspase9 | \nArabidopsis thaliana | \n332191328 | \n
Type II metacaspase | \nPicea abies | \n328887884 | \n
Type II metacaspase | \nMedicago truncatula | \n357451305 | \n
Type II metacaspase | \nTriticum aestivum | \n267850617 | \n
Latexprotein | \nHevea brasiliensis | \n4235430 | \n
Type I metacaspase | \nSaccharomyces cerevisiae | \n151945290 | \n
Type I metacaspase | \nSchizosaccharomyces pombe | \n19076003 | \n
List of proteins utilized on the analysis of sequence alignment
The p20 and p10 domains of the G. max metacaspase were confronted to the Protein Data Bank (http://www.wwpdb.org/) for the search of templates for molecular prediction employing the software Swiss Model [84] (http://swissmodel.expasy.org/). The visualization, the analysis, the validation and the improving of the protein structures were performed with the assistance of the software NOC [85] (http://noch.sourceforge.net).
The initial alignment of caspases and metacaspases sequences with the whole protein sequences (Figure 3) divided them into two groups: sequences with larger pro‐domains and shorter loops; and sequences with smaller pro‐domains and larger loops. Using the position of the catalytic dyad His‐Cys as guide and after the removal of pro‐domains and loops, the core of p20 and p10 domains became evident. The number of amino acid residues counted 235 residues, being 146 from p20 and 85 from p10 domain. It was also possible to underline the approximate segment borders for the domains of the G. max metacaspase, and it was seen that, besides the conserved catalytic dyad position, their adjacent residues (on primary sequence) shared the same chemical nature, encompassing all the protein sequences, despite the phylogenetic distance. The same was seen for the residues close to these regions, considering the p20 segment. In relation to p10, its catalytic residues presented conserved position, despite the chemical divergence (Asp for metacaspases and Arg for caspases). This segment also revealed conservation along its extension, such as the Ser residues close to the catalytic site, amino acids with shorter lateral groups and polar residues. Concerning the prediction of secondary structure organization, it was possible to determine that about 33% of the sequence can form α‐helix/3‐10 helix and that about 23% can take part of β‐sheet conformation (Figure 3).
Sequence alignment of caspases and metacaspases, displaying the corresponding secondary structure. Blue: catalytic histidine; yellow: catalytic cysteine; brown: polar residues; grey: aliphatic residues; pink: contact aspartic acid and arginine residues; green: hydrophobic residues. Blue box: β conformation; red box: α‐helix/310helix. Star: catalytic residues.
The search for a protein that could be applied as a template using the individual subunits from G. max metacaspase, resulted in only one choice with a significant sequence similarity rate for the p20 subunit: the chain A of a protein complex from Geobacter sulfureduccens (PDB‐3BIJ). The identity and similarity rates between the template (from the 60th to the 148th residue) and p20 were of 35 and 55%, respectively. As the analysis of tertiary structure of the subunits and the total protein sequences were compatible, even with the high primary structure difference, 3BIJ was used as a template for the construction of a structural model for metacaspase 4. Only one α‐helix from 3BIJ was removed for adjustment to the target protein sequence.
\nConcerning the established structural model (Figure 4), it was possible to note that the amino acids residues from the catalytic dyad (His/Cys) of G. max metacaspase were closer arranged, spatially. The contact Asp residue also kept this position, suggesting that they are, in fact, intimately associated to the enzymatic catalysis. The model also presented a tridimensional structure close to that of caspases and related proteins, with a core of β‐conformations originated from both p20 and p10 being encircled by α‐helixes, also originated from both domains. It totalized two β‐conformations on its central position which are originated from p10, while p20 contributed with tree of these secondary structures. Other two β‐sheets occupied the model extremity. With relation to the α‐helixes and 3–10 helixes, there are eight of those that encircled the β‐conformation core. Three of those are originated from p20; other two are from p10. The remaining helixes are disposed on the model extremities.
Structural model generated for metacaspase 4 ofGlycine max. (A) Lateral and (B) top view from catalytic site. The p20 and p10 domains are colored in green and red respectively. The lateral chains from the catalytic amino acids are in evidence.
The sequence comparison of metacaspases and caspases domains p20 and p10 clearly shows differences on the amino acid composition and disposition. Nevertheless, the segments, once aligned, displayed conserved positions of catalytic residues and of other amino acid residues with conserved physical‐chemical properties, what is important to the arrangement on a similar secondary structure. Among the p20 secondary structure, six peptide sequences participate on α‐helixes and 3–10 helixes, and seven composes β‐conformations; for p10, three sequences form helix structures and two originate β‐conformations. Together, these structures are organized in a similar way to that observed for the protein from the CD clan of C14 family of the cysteine proteases, which includes caspases and metacaspases [86]. The constructed G. max metacaspase model shows that these sequences are organized in form to present a core of β conformations encircled by five helixes, with the amino acid residues which compose the active site localized in one of the enzyme central axis poles, out of the β‐sheets and α‐helixes region.
\nThe used template here was the chain A of the protein not functionally uncharacterized 3BIJ protein of G. sulfureduccens. This is the same protein used by Dudkiewicz and Piszczek [87], for the prediction of a model for Triticum aestivum type II metacaspase. Interestingly, 3BIJ was seen to be a better template for the considered metacaspase than the Homo sapiens caspase 7, whose similarity with soybean metacaspase was also high.
\nCuriously, a number of recent reports have demonstrated a difference of cleavage specificity among caspases and metacaspases. The recombinant metacaspase McIIPa, from P. abies, was shown to be efficient on the cleavage of the peptide sequence EGR and GRR, but not of VEID and YVAD, which are processed by caspases [46]. In 2008, He et al. [88] demonstrated that the recombinant metacaspase 8 from Arabidopsis thaliana was efficient on the cleavage of the sequence GRR, being unable to process DEVD, VEID, IETD and YVAD. This difference on enzymatic activity nature of metacaspases and caspases are generating an open discussion on literature. The denomination “caspase” itself gives clues to these discussions concerning to the particularities presented by caspases and metacaspases. Carmona‐Gutierrez et al. [51] suggested that metacaspases and caspases present characteristics that fulfil the homology criteria, as they participate of a common program, share substrates and by the fact that metacaspase genes are present in all organisms, except superior animal taxa. In this scenario, the caspase genes could be derived from metacaspases.
\nIn response, Enoksson and Salvesen [52] defended that yeasts and plants would employ PCD programmes other than apoptosis, what would be an innovation when compared to animals. Also they argue that, even if metacaspases and caspases share the tridimensional structure, the cleavage specificity displayed by them could show that they are derived from a common ancestor, which was neither caspase nor metacaspase.
\nThis scenery is reinforced by data from the work of Koonin and Aravind [11], which showed that metacaspases have similarities with α‐proteobacteria homologues, the group of endosymbiotic mitochondria ancestors, being the metacaspases from prokaryotic origin. Also, it was demonstrated that bacterial homologues of caspase‐related proteins showed a greater diversity of phyletic distribution, domain architecture and sequence than their eukaryotic counterparts, suggesting that events of gene transference from prokaryote to eukaryotes could be an explanation for the distribution of caspase‐related genes, what could have been assured by multiple bacterial gene infusions [87].
Please provide page range for Ref. [29].
--\x3eactivation and function. Cold Spring Harbor Perspectives in Biology. 2013;At the present time, vacuum plasma methods for the formation of multicomponent thin-film structures based on magnetron or vacuum arc gas discharges are widely used in the industry. These methods allow to obtain a wide class of functional coatings, such as optical, hardening, anticorrosion, antibacterial, etc. A particular case of the known vacuum plasma methods of coating formation is ion-assisted deposition [1, 2, 3, 4, 5, 6, 7, 8]. The method involves continuous or periodic bombardment of growing thin films by accelerated ions.
\nThe results of the bombardment of the substrate and the growing films by accelerated ions are [1, 2, 3, 4, 5, 6, 7, 8]:
cleaning of the substrate surface and removing traces of water and hydrocarbons;
increase of density and, in some cases, modification of the growing film structure;
removal of loosely bounded molecules during the film growth;
improving the adhesion of the film to the substrate;
better control of the film stoichiometric composition in the case of chemically active gas usage.
The concept of ion-stimulated deposition was proposed more than 70 years ago by Berghaus [1]. More than two decades later, the technology was implemented in the experimental works by Mattox and MacDonald [2], as well as by Mattox and Kominiak [3].
\nCurrently, vacuum deposition of films is carried out by way of thermal evaporation, magnetron, vacuum arc, and ion-beam deposition [4, 5, 6, 7, 8]. Bombardment of substrates and growing films is implemented using accelerated ion beams, generated in ion sources or gas-discharge plasma. In the latter case, a negative bias is applied to the substrate, which accelerates the ions in the direction perpendicular to the surface of the growing film.
\nThe effect of the ion flux treatment on the thin-film properties substantially depends on the flux magnitude, energy and mass of the ions, as well as the ratio between the flows of the assisting ions and the atoms of the deposited substance. The question of the optimal magnitude of energy, which has to be introduced into the growing film per a single deposited atom, was examined in [5, 9, 10, 11, 12].
\nIn [10], the results of numerous experiments on the deposition of coatings using ion beam stimulation are analyzed. It is shown that the most significant changes in the properties of deposited films occur, when each of the deposited atom obtains additional energy in the range of 1.0–100 eV. However, it was shown in [11] that in the case of Ti-Al-N film deposition, composition of the film, predominant orientation, and distances between atoms in the lattice significantly depend on whether the energy is introduced by one high-energy ion or by several low-energy ions. As shown in [12], the problem is that in the above cases the energy is transferred to the deposited atoms irregularly. High-energy ions are capable of creating defects in the deeper layers of the coating and the substrate; having been reflected from the surface of the substrate, the ions acquire energy, which significantly depends on the substrate material. This results in the fact [12] that the energy required for the film growth at the initial stage may greatly differ from the energy, which is required at the final growth stage, when the assisting ions interact only with the atoms of the deposited substance. It should be noted that when the ion energy exceeds 15–30 eV [13], the growing film can be sputtered with a beam of fast ions. The use of ion beams with energies up to 30 eV makes it possible to reduce these effects to a minimum.
\nThe use of vacuum plasma methods of forming functional coatings in the industry requires the ensuring of high-speed film deposition and, accordingly, the use of significant fluxes of assisting ions. Literature review [6, 13] shows that the ion fluxes generated by gridded and gridless ion beam sources are often insufficient to assist the growth of films produced using magnetron and vacuum arc methods. Besides, there is a complex problem of matching the operation of gas-discharge sources generating assisting ions and coating systems in terms of pressure.
\nIn [14], the possibility of generating plasma flows with the ion component density of up to 20–30 mA/cm2 and independently controlled ion energy within the range of 20–120 eV by using a combination of arc discharge with inductive radio-frequency (RF) discharge in external magnetic field is shown. The results obtained in [14] served as the starting point for the development of a plasma reactor [15, 16, 17, 18], intended for the magnetron sputtering of functional coatings using stimulation by ions, generated in RF inductive discharge with external magnetic field.
\nThe task of developing a plasma reactor for magnetron sputtering deposition of ion-stimulated coatings has determined the composition of the reactor: one or more magnetron sources to provide the flow of sputtered particles onto a substrate and gas-discharge source, which provides the generation of a magnitude-controlled flux of ions, bombarding the substrate. The ion flux energy control is provided by a direct current (DC) or RF biasing of the substrate.
\nTo enable successful operation of the plasma reactor, it is necessary to ensure that:
The working areas of the magnetron and gas-discharge sources are matched in terms of pressure.
Changing of external parameters of the gas-discharge source ensures coordination of the flux of assisting ions and the flux of sputtered particles entering the substrate.
Changing of external parameters of the gas-discharge source ensures uniform density of ion current density on the substrate not worse than 10%.
The plasma reactor operates using both inert and chemically active gases.
Let us do some estimates. Taking into account typical rate of magnetron sputtering, it is advisable to ensure that the density of the stimulating ion current varies within the range of 0.1–3 mA/cm2. This means that the plasma density must vary within the range of 0.03–1 × 1011 cm−3.
\nA literature review [19, 20, 21] has shown that it is reasonable to consider two modifications of inductive RF discharge as the options for implementing the working process in the gas-discharge source, namely, inductive RF discharge in the absence and in the presence of external magnetic field. In the first case, the magnitude of the assisting ion flow will be determined by the power of the RF power supply. In the second case, two external parameters—the power of the RF power supply and the induction of an external magnetic field—will allow to control the values of plasma parameters and their spatial distribution in the plasma reactor.
\nIn modern industrial installations, the diameter of substrates usually exceeds 200 mm. Large diameter of the plasma reactor makes it difficult to generate strong magnetic fields in the volume of the reactor. It is known [22, 23, 24, 25] that when an external magnetic field with induction of less than 100 Gs is imposed on inductive RF discharge, a peak of electron density is observed at certain resonant values of the magnetic field. The absolute values of plasma density are close to those required in case of our task. The physically observable density peak is associated with resonant excitation in the plasma reactor of helicon and Trivelpiece-Gould waves [22, 23, 24, 25]. Provided that the pressure in the reactor does not exceed 10 mTorr, the Trivelpiece-Gould wave is a bulk wave, penetrates deep into plasma, and determines the absorption of RF power [24, 25].
\nIn [26], the authors offered a scheme of two-chamber process plasma source, based on inductive RF discharge with external magnetic field, diverging toward the substrate. Following the scheme [26], preliminary experiments on the study of the discharge parameters have been carried out with the plasma reactor having two chambers [27]. The model consisted of two cylindrical quartz chambers of different diameters and a magnetic system. The upper part of the reactor (gas-discharge chamber) had a diameter of 10 cm and a height of 25 cm. The lower part of the reactor (process chamber) had 46 cm diameter and 30 cm height. The RF power input unit—a three-turn solenoid antenna—was located on the outer surface of the gas-discharge chamber. The magnetic system allowed generation in the volume of the process chamber of uniform magnetic field, converging and diverging toward the lower flange where the substrate was located.
\nExperiments have shown that when using an inductive RF discharge in the absence of magnetic field, the discharge is concentrated in the gas-discharge chamber, while the plasma density in the process chamber near the substrate is vanishingly small. A different situation is observed when using external magnetic field, diverging in the region of the gas-discharge chamber and uniform in the region of the process chamber. Under argon pressures, when the electron mean free path exceeds the longitudinal size of the system, the imposition of magnetic field results in significant changes in the discharge length [27]. In the absence of magnetic field, the discharge is concentrated in the gas-discharge chamber. An increase in the magnitude of the magnetic field first results in the appearance of plasma in the upper part of the technological chamber. Then, the length of the intensely glowing part of the discharge in the process chamber starts to grow, and, finally, the discharge closes at the bottom flange. The movement of electrons across the magnetic field is difficult; therefore, an extended plasma column, sharply outlined in the radial direction, appears in the process chamber. In parallel with the increase in the discharge length near the substrate, an increase in the probe ion saturation current is observed.
\nFigure 1 shows variation of the axial distribution of the probe ion saturation current when the magnetic field induction grows. In the absence of magnetic field, the discharge is concentrated in the upper part of the plasma source; as the magnetic field increases, the ion current in the lower chamber gets increased. When the magnetic field exceeds 36 Gs, the discharge gets localized in the process chamber of the reactor.
\nProbe ionic saturation current distribution along z axis of plasma source depending on external magnetic field magnitude: 0.7 mTorr, 400 W, and 13.56 MHz.
This effect is observed for all of the examined powers of the RF power supply, and the maximum values of the ion current are increasing in proportion to the input power. The achieved plasma concentrations near the substrate correspond to those required in this work. Detailed studies of the physical causes for the axial redistribution of plasma density as the magnetic field induction increases have shown that the effect is associated with the patterns of excitation of partial standing waves in the plasma [17, 27]. It should be noted that the use of other configurations of the magnetic field did not result in significant increase in the ionic saturation current near the substrate.
\nThe obtained results served as the basis for the development of a semi-industrial installation for magnetron deposition of coatings with ion assistance.
\nPreliminary results, obtained using a plasma reactor prototype, have served as the basis for a semi-industrial installation [16, 17, 18]. Diagram of this prototype is shown in Figure 2. The reactor consists of two parts. The main part is a metal cylindrical process chamber having 500 mm diameter and 350 mm height. At the bottom of the chamber, there is a rotating table for the accommodation of samples being treated. To facilitate spectrometric studies of plasma parameters, two optical inspection windows are located above the table right opposite to each other. The magnetron source is installed on the side surface of the process chamber.
\nPlasma reactor prototype diagram: (1, 2) gas-discharge and process chambers, (3) antenna, (4, 5) top and bottom electromagnets, (6) magnetron, (7) rotating table, and (8) window.
Quartz gas-discharge chamber having 250 mm length and 220 mm diameter is mounted on top of the process chamber. From above the chamber, volume is covered with a glass blind flange; from beneath the chamber is sealed with a metal flange with an opening that allows plasma to penetrate into the main chamber.
\nBelow the process chamber, a pumping system is installed, consisting of rotary and turbo molecular pumps. The working gas is supplied to the reactor through the gas inlet located in the upper part of the gas-discharge chamber.
\nThe magnetic system consists of two electromagnets, located in the upper and lower parts of the process chamber. The electromagnets ensure generation of diverging magnetic field in the area of the gas-discharge chamber. The magnitude of the magnetic field in each specific point in the volume of the process chamber is determined by the currents Itop, Ibot flowing through the top and bottom electromagnets, respectively, and one and the same value of magnetic induction can be provided by setting different ratios between the currents of the magnets. In this regard, in the further section, the dependence of the discharge parameters on Itop, Ibot is given in the graphs illustrating the results of experiments. Table 1 contains the magnetic field induction values achieved near the antenna, in the center of the process chamber and near the substrate at different values of Itop, Ibot.
\nTop magnet current (Gs) | \nBottom magnet current (Gs) | \nMagnetic field in the substrate plane (Gs) | \nMagnetic field in the bottom part of the gas-discharge chamber (Gs) | \n
---|---|---|---|
3 | \n5 | \n39 | \n49 | \n
4 | \n5 | \n40 | \n63 | \n
5 | \n5 | \n42 | \n78 | \n
7 | \n5 | \n45 | \n107 | \n
3 | \n1 | \n11 | \n45 | \n
3 | \n3 | \n25 | \n47 | \n
3 | \n7 | \n53 | \n50 | \n
3 | \n9 | \n66 | \n52 | \n
3 | \n12 | \n87 | \n55 | \n
10 | \n4 | \n42 | \n150 | \n
1 | \n6 | \n42 | \n20 | \n
Correlation between the currents of the magnets and the magnitude of the magnetic field near the substrate and in the gas-discharge chamber.
To excite an inductive RF discharge, a solenoid antenna is used, which is located on the outer surface of the quartz chamber. The ends of the antenna are connected through the matching system to the RF power source, having the operating frequency of 13.56 MHz and an output power of up to 1000 W. In the inductive RF discharge, the power of RF power sources is coupled not only to plasma but is wasted in the antenna too. In order to measure the RF power value Ppl coupled to plasma, the method described in [24] was used.
\nTo ignite and maintain the magnetron discharge, the RF power source with the operating frequency of 13.56 MHz and output power up to 1000 W is used.
\nTo study the homogeneity of plasma near the substrate, a movable Langmuir probe was used, capable of traveling along the diameter of the substrate. When measuring the dependence of the probe ionic saturation current on the magnitude and configuration of the magnetic field, 60 V potential negative in respect of to the walls of the main chamber is applied to the probe.
\nIn parallel with probe measurements, the plasma glow spectrum was studied. Plasma radiation via an optical fiber was fed to the input of the MDR-40 monochromator, on the output of which photomultiplier FEU-100 was installed. The signal from the photomultiplier was amplified and fed to an ADC connected to a computer. The spectrum was scanned in the wavelength range of 400–700 nm. In addition to the glow spectrum, longitudinal distribution of the spectral lines intensity was measured. To this end, the light guide was moved along the generating line of the process chamber within the diagnostic window opening.
\nThin films were deposited onto silicon substrates when only the magnetron was operating and when the magnetron and the gas-discharge plasma source were operating together in the absence and in the presence of bias on the substrate. The morphology of the surface and the cleavage of the films were studied with the help of scanning electron microscope Supra-40. The electro-physical characteristics of the coatings were measured with the help of a two-probe method.
\nAt the first stage of the study, the parameters of the plasma generated by inductive RF discharge were studied depending on the size and configuration of the external magnetic field, the power of the RF power supply, and the pressure of argon in the absence of the magnetron discharge.
\nExperimental studies of the discharge have shown that when a longitudinal magnetic field is imposed on the discharge, an extended plasma column is formed in the process chamber, similar to that observed in the prototype reactor. The diameter of the plasma column is approximately equal to the diameter of the quartz chamber that is 20 cm (Figure 3).
\nPhoto of the discharge in the plasma reactor.
As with the prototype, a change in the magnetic field made it possible to control the longitudinal distribution of plasma density. Figure 4 shows correlation between the plasma glow intensity I1, measured in the central part of the process chamber, and at the substrate I3. An increase in the currents through the magnet, located near the gas discharge Itop and the substrate Ibot, results in equalization of the distribution of the plasma glow intensity along the discharge axis. This confirms the conclusion about the formation of the plasma “column,” closing on the substrate.
\nThe ratio of plasma glow intensity near the substrate I3 to the plasma glow intensity near the magnetron I1 versus the current flowing through the bottom electromagnet. Corresponding values of the current through the top electromagnet are shown in the menu in the figure. Pressure, 0.2 Pa.
Figures 5 and 6 show the dependence of the portion of the power Ppl coupled to the argon plasma, on the values of the currents Itop, Ibot through the top and bottom magnets, provided that the power of the RF power supply is 1000 W. As can be seen, Ppl is determined by the magnetic field B, which is generated by the current through the top electromagnet located in the area of the gas-discharge chamber. The effect induced by the bottom magnet is noticeable at low currents of the top magnet, when contribution of the bottom electromagnet to the values of the magnetic field induction is significant. The non-monotonic character of dependence of the absorbed power on B is also worth noticing. Ppl reaches the maximum in the Itop range between 3 and 5 A, which corresponds to magnetic fields in the area of the gas-discharge chamber of 50–80 Gs. The non-monotonous nature of the dependence of the absorbed power on the external magnetic field is retained when argon is replaced by other working gases, such as neon, argon, and oxygen. However, the position of the energy input maximum significantly depends on the working gas used. Thus, with the use of oxygen, position of the Ppl(B) maximum at the RF power supply power of 1000 W is reached at Itop = 2 A (see Figure 6).
\nDependence of the portion of power, coupled to plasma, on the values of currents through the top and bottom magnets, provided that the RF power supply power is 1000 W. Argon pressure, 0.7 Pa.
Dependence of the portion of power, coupled to plasma, on the values of currents through the top and bottom magnets, provided that the RF power supply power is 1000 W. Oxygen pressure, 0.7 Pa.
Figure 7 shows radial dependence of the RF longitudinal magnetic field Bz, measured at various Itop values. One can see that Bz under condition of the best absorption of the RF power (at the magnetic field of about 80 G in the area of the gas-discharge chamber) reaches its maximum on the discharge axis, that is, the RF fields penetrate deep into the plasma (see Figure 7). This indicates excitation of bulk waves in the discharge.
\nRadial dependence of the RF longitudinal magnetic field.
Calculation of the dependence of the RF fields, excited in plasma, on the external magnetic field, made on the basis of the theoretical model of a limited inductive plasma source with external magnetic field [25], has shown that interconnected helicons and Trivelpiece-Gould waves are excited in the discharge under considered experimental conditions. The Trivelpiece-Gould wave is a bulk wave, and it is the dissipation of its energy that determines the absorption of RF power in the plasma.
\nThe conclusion concerning the resonant excitation of bulk waves at the current of the top magnet of 5A is also confirmed by measurements of the probe ionic saturation current near the substrate (see Figure 8). An increase in the current of the top magnet within the range from 1 to 5 A results in increase in the absolute values of the ionic current and the formation of a bell-shaped distribution. As the current is further increased to 7 A, the portion of RF power absorbed by the plasma and the absolute value of the probe ionic saturation current decrease. It is necessary to note the result that is important for technological applications: at the top magnet current of 3A, the most uniform distribution of the ion current is observed. Deviation of the ionic current values from the average ones within the 20 cm diameter does not exceed 10%. Remarkably, the most uniform radial distribution near the substrate is observed not in the resonance region, but when approaching the resonance region.
\nRadial dependence of ionic saturation current at different top magnet currents while the bottom magnet current is fixed.
The most uniform distribution, obtained at the top magnet current of 3A, corresponds to the ionic current density of about 1 mA/cm2. This value may be insufficient for ionic assistance at high rates of film deposition. Additional experiments have shown that increasing the bottom magnet current allows to increase the ionic current density by two to three times and to obtain homogeneous plasma within the diameter of 15 cm (Figure 9, curves 1 and 2).
\nRadial dependence of the ionic saturation current at different bottom magnet currents while the top magnet current is fixed.
Figure 9 additionally shows the radial distributions of the ionic saturation current, obtained at the top magnet current of 1A. As a reminder, the point Itop = 1 A is located before the maximum of the Ppl(Itop) curve. It is natural to assume that at this point the RF fields do not yet penetrate the main volume of the plasma. Taking into account that the movement of electrons across the magnetic field is difficult, the distribution of the ionic current with a dip on the axis is observed.
\nLet us further examine the results of the probe studies in more detail. A typical probe characteristic is shown in Figure 10.
\nDependence of the probe electron current on the probe potential.
As can be seen, the electron energy distribution is close to Maxwell one. Figure 11 shows the dependence of electron density in the substrate region on the values of currents through the top and bottom magnets at fixed values of current on the top and bottom magnets, respectively.
\nDependence of electron density in the substrate region on the values of currents through the top (a) and bottom (b) magnets at fixed values of current on the top and bottom magnets, respectively.
As can be seen, the range of changes in plasma density corresponds to that which is necessary for the successful implementation of ion assistance technology as part of the magnetron sputtering.
\nAdditional experiments have shown that the best argon pressure range in terms of technological applications is the range between 0.5 and 0.7 Pa. At lower pressures, it is not possible to obtain the required uniformity of the ion current, while an increase in pressure to 1.5 Pa leads to a drop in the values of the ion current.
\nAt the next stage of the work, the plasma parameters were studied during the simultaneous operation of both the magnetron discharge and inductive RF discharges with external magnetic field. Experiments have shown that the joint operation of magnetron and inductive RF discharges results in a decrease in the threshold pressure, at which magnetron is capable of operating. Titanium spectral lines appeared in the glow spectrum of the magnetron discharge only and at the argon pressure of 0.7 Pa. When magnetron and RF inductive discharges were operating together, the plasma glow spectrum was enriched with titanium lines already at the pressure of 0.3 Pa.
\nFigure 12 shows the current-voltage characteristics of the magnetron operating at DC mode both independently and together with the gas-discharge source. As can be seen, the sputtering apparatus is capable of operating at substantially lower voltages applied to the cathode.
\nCurrent-voltage characteristics of the magnetron operating independently (black curve) and together with the inductive RF discharge with magnetic field (red curve).
To test the effect of ionic stimulation on the properties of functional coatings, thin films were deposited using the magnetron only and using the magnetron and RF sources together with and without applying an additional bias to the substrate. Coatings made of the following materials were used as test samples: Ti, Al, SiAl, SiO2, and C.
\nThe experiments involving deposition of titanium coatings were aimed at the studying of the effects of the assisting ionic flux magnitude on electro-physical properties of films and their microhardness. It was shown that increase in the power of RF power supply connected to antenna in the range from 0 to 500 W results in almost twofold increase in specific resistivity of the films; at the same time, the microhardness of the films increases by 25%. The observed changes are obviously the result of a change in the film structure. Titanium film morphology studies have shown that the irradiation of films with a flux of accelerated ions leads to a slight decrease in the grain size in the structure of the films. Application of a DC bias to the substrate was accompanied by the smoothing of the surface of films.
\nThe reduction of grain size in the structure of films in the presence of stimulating ion flux is most clearly seen in the film surface images and the images of aluminum film cleavages, shown in Figure 13.
\nAluminum film surface (a and c) and cross-section (b and d).
Interesting results were obtained while depositing silicon with 10% aluminum content. The sample film surface morphology and cleavage are shown in Figures 14–16. As can be seen, the samples treated using ionic assistance acquire a columnar structure. When a bias is applied to the substrate, the columnar structure grows at an angle to the normal.
\nFilm surface and cross-section: Si (Al 10%) deposition, without ionic assistance.
Film surface and cross-section: Si (Al 10%) magnetron deposition with ionic assistance without applying a bias to substrate.
Film surface and cross-section: Si (Al 10%) magnetron deposition with ionic assistance plus a bias applied to the substrate.
It is known that the ionic stimulation results in significant change in the electro-physical properties of carbon films [28]. Figure 17 shows the dependence of resistance of a various series of carbon films on the bias voltage [29]. As can be seen, there is a sharp increase in the resistance of the films at the assisting ion energy of 45 eV.
\nDependence of the ratio of film resistance to its thickness on the bias Ucm applied to its substrates.
The results of the study of plasma parameters in a plasma reactor based on a combination of magnetron and magnetically activated RF discharge indicate the promising outlook as regards to industrial application of the innovative facility. Approaching the region of resonant absorption of RF power by optimizing the magnitude and configuration of the external magnetic field makes it possible to obtain a uniform within 10% radial distribution of the ion current across the diameter of 150 mm. When the RF power supply power is 1000 W, the ion current density on the substrate can be adjusted in the range of 0.1–3 mA/cm2. The use of ion assisting results in a fundamental change in the structure and properties of functional coatings, deposited using a magnetron.
\nThe work was carried out under partial financial support of the RFBR grant 16-02-00646.
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