N-terminal Cleavage Site Research Articles

Prediction of mitochondrial targeting signals and their cleavage sites.

In this chapter we survey prediction tools and computational methods for the prediction of amino acid sequence elements which target proteins to the mitochondria. We will primarily focus on the prediction of N-terminal mitochondrial targeting signals (MTSs) and their N-terminal cleavage sites by mitochondrial peptidases. We first give practical details useful for using and installing some prediction tools. Then we describe procedures for preparing datasets of MTS containing proteins for statistical analysis or development of new prediction methods. Following that we lightly survey some of the computational techniques used by prediction tools. Finally, after discussing some caveats regarding the reliability of such methods to predict the effects of mutations on MTS function; we close with a discussion of possible future directions of computer prediction methods related to mitochondrial proteins.

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

Subtilase-mediated biogenesis of the expanded family of SERINE RICH ENDOGENOUS PEPTIDES.

Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2.

Identification of the genes at S and Z reveals the molecular basis and evolution of grass self-incompatibility.

Self-incompatibility (SI) is a feature of many flowering plants, whereby self-pollen is recognized and rejected by the stigma. In grasses (Poaceae), the genes controlling this phenomenon have not been fully elucidated. Grasses have a unique two-locus system, in which two independent genetic loci (S and Z) control self-recognition. S and Z are thought to have arisen from an ancient duplication, common to all grasses. With new chromosome-scale genome data, we examined the genes present at S- and Z-loci, firstly in ryegrass (Lolium perenne), and subsequently in ~20 other grass species. We found that two DUF247 genes and a short unstructured protein (SP/ZP) were present at both S- and Z- in all SI species, while in self-compatible species these genes were often lost or mutated. Expression data suggested that DUF247 genes acted as the male components and SP/ZP were the female components. Consistent with their role in distinguishing self- from non-self, all genes were hypervariable, although key secondary structure features were conserved, including the predicted N-terminal cleavage site of SP/ZP. The evolutionary history of these genes was probed, revealing that specificity groups at the Z-locus arose before the advent of various grass subfamilies/species, while specificity groups at the S-locus arose after the split of Panicoideae, Chloridoideae, Oryzoideae and Pooideae. Finally, we propose a model explaining how the proteins encoded at the S and Z loci might function to specify self-incompatibility.

Characterization of Calpain and Caspase-6-Generated Glial Fibrillary Acidic Protein Breakdown Products Following Traumatic Brain Injury and Astroglial Cell Injury.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament III protein of astroglia cells which is upregulated in traumatic brain injury (TBI). Here we reported that GFAP is truncated at both the C- and N-terminals by cytosolic protease calpain to GFAP breakdown products (GBDP) of 46-40K then 38K following pro-necrotic (A23187) and pro-apoptotic (staurosporine) challenges to primary cultured astroglia or neuron-glia mixed cells. In addition, with another pro-apoptotic challenge (EDTA) where caspases are activated but not calpain, GFAP was fragmented internally, generating a C-terminal GBDP of 20 kDa. Following controlled cortical impact in mice, GBDP of 46-40K and 38K were formed from day 3 to 28 post-injury. Purified GFAP protein treated with calpain-1 and -2 generates (i) major N-terminal cleavage sites at A-56*A-61 and (ii) major C-terminal cleavage sites at T-383*Q-388, producing a limit fragment of 38K. Caspase-6 treated GFAP was cleaved at D-78/R-79 and D-225/A-226, where GFAP was relatively resistant to caspase-3. We also derived a GBDP-38K N-terminal-specific antibody which only labels injured astroglia cell body in both cultured astroglia and mouse cortex and hippocampus after TBI. As a clinical translation, we observed that CSF samples collected from severe human TBI have elevated levels of GBDP-38K as well as two C-terminally released GFAP peptides (DGEVIKES and DGEVIKE). Thus, in addition to intact GFAP, both the GBDP-38K as well as unique GFAP released C-terminal proteolytic peptides species might have the potential in tracking brain injury progression.

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo–N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

Gold Nanoparticles Augment N-Terminal Cleavage and Splicing Reactions in Mycobacterium tuberculosis SufB.

Protein splicing is a self-catalyzed event where the intervening sequence intein cleaves off, joining the flanking exteins together to generate a functional protein. Attempts have been made to regulate the splicing rate through variations in temperature, pH, and metals. Although metal-regulated protein splicing has been more captivating to researchers, metals were shown to only inhibit splicing reactions that confine their application. This is the first study to show the effect of nanoparticles (NPs) on protein splicing. We found that gold nanoparticles (AuNPs) of various sizes can increase the splicing efficiency by more than 50% and the N-terminal cleavage efficiency by more than 45% in Mycobacterium tuberculosis SufB precursor protein. This study provides an effective strategy for engineering splicing-enhanced intein platforms. UV-vis absorption spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy (TEM) confirmed AuNP interaction with the native protein. Quantum mechanics/molecular mechanics (QM/MM) analysis suggested a significant reduction in the energy barrier at the N-terminal cleavage site in the presence of gold atom, strengthening our experimental evidence on heightened the N-terminal cleavage reaction. The encouraging observation of enhanced N-terminal cleavage and splicing reaction can have potential implementations from developing a rapid drug delivery system to designing a contemporary protein purification system.

Structure of a Cell Entry Defective Human Adenovirus Provides Insights into Precursor Proteins and Capsid Maturation

Maturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310–397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4–96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities.

Enzymatically releasable polyethylene glycol – host defense peptide conjugates with improved activity and biocompatibility

Host defense peptides (HDPs) have been the subject of great interest for the treatment of multidrug-resistant bacterial infections due to their multimodal activity and low induction of resistance. However, aggregation, toxicity, and short biological half-life have limited their applicability for clinical treatment. Many methods have been explored to alleviate these issues, such as polymer (e.g., polyethylene glycol (PEG)) conjugation, but these are often accompanied by reductions in the activity of the HDP. Here, we detail the design of a novel PEG-HDP conjugate incorporating an enzymatic cleavage sequence targeting matrix metalloproteinases (MMPs) that accumulate at sites of inflammation and infection. Addition of the cleavage sequence onto either the N- or the C-terminal region of the parent peptide (peptide 73, a derivative of the HDP aurein 2.2) was explored to determine the location for optimal antimicrobial activity following MMP cleavage; furthermore, the susceptibility of the peptide to MMP cleavage after conjugation to 2 kDa or 5 kDa PEG was examined. The top candidate, L73, utilized an N-terminal cleavage site that was subsequently conjugated to a 2 kDa PEG polymer. Both L73 and the conjugate exhibited no antimicrobial activity in vitro until cleaved by purified MMP, which liberated a peptide fragment with 16- or 63-fold improved activity, respectively, corresponding to a minimum inhibitory concentration (MIC) of 8 μg/mL, comparable to that of peptide 73 (4 μg/mL). Furthermore, PEG conjugation improved the blood compatibility and reduced the aggregation tendency of the HDP in vitro, indicating enhanced biocompatibility. When administered as a single subcutaneous dose (~3.6 mg, or a peptide concentration of 142 mg/kg) in a mouse abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, the conjugate displayed strong activity, reducing abscess size and bacterial load by 73.3% and 58-fold, respectively. This activity was completely lost when the cleavage site was rendered resistant to MMPs by the substitution of two d-amino acids, supporting the hypothesis that antimicrobial activity was dependent on cleavage by MMPs, which were shown here to increasingly accumulate at the abscess site up to 18 h post infection. Finally, the conjugate displayed biocompatibility in vivo, with no identifiable toxicity or aggregation.

β1-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte β1-adrenergic receptors

β1-adrenergic receptors (β1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the β1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the β1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents β1AR N-terminal cleavage, and that N-terminal truncation influences β1AR signaling to downstream effectors. However, the site(s) and mechanism for β1AR N-terminal cleavage in cells was not identified. This study shows that β1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated β1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the β1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the β1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates β1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in β1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte β1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated β1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant β1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the β1AR N-terminus as a structural determinant of β1AR responses that can be targeted for therapeutic advantage.

Molecular docking between human TMPRSS2 and SARS-CoV-2 spike protein: conformation and intermolecular interactions.

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational attributes of the molecular complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike protein. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provides structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

Molecular characterisation of atypical BSE prions by mass spectrometry and changes following transmission to sheep and transgenic mouse models.

The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts.

Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants

We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR.

A Single Maturation Cleavage Site in Adenovirus Impacts Cell Entry and Capsid Assembly.

Proteolytic maturation drives the conversion of stable, immature virus particles to a mature, metastable state primed for cell infection. In the case of human adenovirus, this proteolytic cleavage is mediated by the virally encoded protease AVP. Protein VI, an internal capsid cement protein and substrate for AVP, is cleaved at two sites, one of which is near the N terminus of the protein. In mature capsids, the 33 residues at the N terminus of protein VI (pVIn) are sequestered inside the cavity formed by peripentonal hexon trimers at the 5-fold vertex. Here, we describe a glycine-to-alanine mutation in the N-terminal cleavage site of protein VI that profoundly impacts proteolytic processing, the generation of infectious particles, and cell entry. The phenotypic effects associated with this mutant provide a mechanistic framework for understanding the multifunctional nature of protein VI. Based on our findings, we propose that the primary function of the pVIn peptide is to mediate interactions between protein VI and hexon during virus replication, driving hexon nuclear accumulation and particle assembly. Once particles are assembled, AVP-mediated cleavage facilitates the release of the membrane lytic region at the amino terminus of mature VI, allowing it to lyse the endosome during cell infection. These findings highlight the importance of a single maturation cleavage site for both infectious particle production and cell entry and emphasize the exquisite spatiotemporal regulation governing adenovirus assembly and disassembly. Postassembly virus maturation is a cornerstone principle in virology. However, a mechanistic understanding of how icosahedral viruses utilize this process to transform immature capsids into infection-competent particles is largely lacking. Adenovirus maturation involves proteolytic processing of seven precursor proteins. There is currently no information for the role of each independent cleavage event in the generation of infectious virions. To address this, we investigated the proteolytic maturation of one adenovirus precursor molecule, protein VI. Structurally, protein VI cements the outer capsid shell and links it to the viral core. Functionally, protein VI is involved in endosome disruption, subcellular trafficking, transcription activation, and virus assembly. Our studies demonstrate that the multifunctional nature of protein VI is largely linked to its maturation. Through mutational analysis, we show that disrupting the N-terminal cleavage of preprotein VI has major deleterious effects on the assembly of infectious virions and their subsequent ability to infect host cells.

Structure mediation in substrate binding and post-translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase.

Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the β-subunit with cysteine (Serβ1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Serβ1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 Å, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N-terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serβ1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Argα145, Pheα146, and Pheβ24 at the substrate binding pocket. Three conformational transitions of Argα145 and Pheα146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.

Context-Dependent Regulation of Collagen XVII Ectodomain Shedding in Skin

Pemphigoid is a common autoimmune blistering disorder in which autoantibodies target transmembrane collagen XVII (COL17), a component of hemidesmosomes in basal keratinocytes. The ectodomain of COL17 can be cleaved from the cell surface within the juxtamembranous extracellular NC16A domain, and, interestingly, certain autoantibodies of pemphigoid patients preferentially react with the shed ectodomain. These findings suggest that COL17 ectodomain shedding generates neoepitopes on the shed form; however, the regulatory mechanism of the shedding in in vivo skin and the pathogenicity of the neoepitope-targeting antibodies still are uncertain. To address these issues, we produced rabbit antibodies specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. The antibodies showed that certain amounts of the human COL17 ectodomain are cleaved physiologically at Gln(525) in in vivo skin. In contrast, migrating human keratinocytes cleave COL17 at Leu(524) but not at Gln(525). The passive transfer of antibodies reacting with an N-terminal cleavage site of the mouse COL17 ectodomain into neonatal wild-type mice failed to induce blister formation, even though the antibodies bound to the dermal-epidermal junctions, indicating that cleavage site-specific antibodies have reduced or absent pathogenicity for blister formation. This study shows the ectodomain shedding of COL17 to be a physiological event in in vivo human skin that probably generates nonpathologic epitopes on the cleavage sites.

The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (Bc) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. Bc individuals exhibit circulating black spots but fail to melanize upon injury. Although Bc is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing Bc remained unknown. Here, we identified a single point mutation in the pro-phenoloxidase 1 (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1A480V transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype.

A recommended numbering scheme for influenza A HA subtypes.

Comparisons of residues between sub-types of influenza virus is increasingly used to assess the zoonotic potential of a circulating strain and for comparative studies across subtypes. An analysis of N-terminal cleavage sites for thirteen subtypes of influenza A hemagglutinin (HA) sequences, has previously been described by Nobusawa and colleagues. We have expanded this analysis for the eighteen known subtypes of influenza. Due to differences in the length of HA, we have included strains from multiple clades of H1 and H5, as well as strains of H5 and H7 subtypes with both high and low pathogenicity. Analysis of known structures of influenza A HA enables us to define amino acids which are structurally and functionally equivalent across all HA subtypes using a numbering system based on the mature HA sequence. We provide a list of equivalences for amino acids which are known to affect the phenotype of the virus.

Predicting Protein N-Terminal Signal Peptides Using Position-Specific Amino Acid Propensities and Conditional Random Fields

Protein signal peptides play a vital role in targeting and translocation of most secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. Consequently, accurate prediction of signal peptides and their cleavage sites is an important task in molecular biology. In the present study, firstly, we develop a novel discriminative scoring method for classifying proteins with or without signal peptides. This method successfully captured the characteristics of signal peptides and non-signal peptides by integrating hydrophobicity alignment and positionspecific amino acid propensities based on the highest average positions. As a result, this method is capable of discriminating proteins with signal peptides at the overall accuracies of 96.3%, 97.0% and 97.2% by leave-one-out jackknife tests on the constructed benchmark datasets for three different organisms, i.e. Eukaryotic, Gram-negative, and Gram-positive respectively. Secondly, we consider the prediction task of signal peptide cleavage sites as a sequence labeling problem and apply Conditional Random Fields (CRFs) algorithm to solve it. Experimental results demonstrate that the proposed CRFs-based cleavage site finding approach can achieve the prediction success rates of 80.8%, 89.4%, and 74.0% respectively, for the secretory proteins from three different organisms. An online tool, LnSignal, is established for labeling the N-terminal signal cleavage sites and is freely available for academic use at http: //www.csbio.sjtu.edu.cn/bioinf/LnSignal.