C-terminal Processing Research Articles

Transglutaminase mediates the hardening of fish egg envelope produced by duplication of factor XIIIA gene during the evolution of Teleostei.

During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening.

Mono- and Bi-specific Nanobodies Targeting the CUB Domains of PCPE-1 Reduce the Proteolytic Processing of Fibrillar Procollagens

The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies.Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.

Effects of truncations in the N- and C-terminal domains of filensin on filament formation with phakinin in cell-free conditions and cultured cells.

Filensin and phakinin are lens fiber cell-specific proteins that constitute the beaded filaments that are critical for maintaining lens transparency. In the Shumiya cataract rat, filensin 94 kDa undergoes N- and C-terminal proteolytic processing to give a transient 50 kDa fragment and a final 38 kDa fragment, just before opacification. To characterize effects of this processing on filensin function, recombinant proteins representing the two filensin fragments, termed Fil(30-416) and Fil(30-369), respectively, were examined. Fil(30-416) lacks the N-terminal 29 amino acids and the C-terminal 248 amino acids. Fil(30-369) lacks the N-terminal 29 residues and the C-terminal 295 residues. In cell-free assembly characterized by electron microscopy, filensin and Fil(30-416) co-polymerized with phakinin and formed rugged, entangled filaments, whereas Fil(30-369) formed only aggregates. In cultured SW-13 and MCF-7 cells expressing fluorescent fusion proteins, filensin and Fil(30-416) co-polymerized with phakinin and formed cytoplasmic sinuous filaments with different widths, while Fil(30-369) gave aggregates. Therefore, while truncation of the N-terminal 29 amino acids did not affect filament formation, truncation of the C-terminal 295 but not the 248 residues resulted in failure of filament formation. These results indicate that the tail B region (residues 370-416) of rat filensin is essential for filament formation with phakinin. Truncation of the tail B region by proteolytic processing in the cataract rat lens might interfere with beaded filament formation and thereby contribute to opacification.

XCP1 cleaves Pathogenesis-related protein 1 into CAPE9 for systemic immunity in Arabidopsis

Proteolytic activation of cytokines regulates immunity in diverse organisms. In animals, cysteine-dependent aspartate-specific proteases (caspases) play central roles in cytokine maturation. Although the proteolytic production of peptide cytokines is also essential for plant immunity, evidence for cysteine-dependent aspartate-specific proteases in regulating plant immunity is still limited. In this study, we found that the C-terminal proteolytic processing of a caspase-like substrate motif “CNYD” within Pathogenesis-related protein 1 (PR1) generates an immunomodulatory cytokine (CAPE9) in Arabidopsis. Salicylic acid enhances CNYD-targeted protease activity and the proteolytic release of CAPE9 from PR1 in Arabidopsis. This process involves a protease exhibiting caspase-like enzyme activity, identified as Xylem cysteine peptidase 1 (XCP1). XCP1 exhibits a calcium-modulated pH-activity profile and a comparable activity to human caspases. XCP1 is required to induce systemic immunity triggered by pathogen-associated molecular patterns. This work reveals XCP1 as a key protease for plant immunity, which produces the cytokine CAPE9 from the canonical salicylic acid signaling marker PR1 to activate systemic immunity.

The inhibitory activity of a new scorpion venom-derived antimicrobial peptide Hp1470 against Gram-positive bacteria

Antimicrobial peptides (AMPs) are a new type of antibiotic and target a variety of microbes, including antibiotic-resistant strains; thus, AMPs have attracted widespread interest. Scorpion venoms contain many bioactive peptides, including AMPs, and have become an important natural resource of peptide-based drugs. Here, the antibacterial peptide gene Hp1470 from the venom of the scorpion Heterometrus petersii was characterized, and its antibacterial activity was determined. The cDNA sequence of Hp1470 is 300 nt in length and contains an open reading frame (ORF) of 207 nt. The ORF was shown to encode 68 amino acid residues, including a signal peptide (23 aa), a mature peptide (13 aa), a C-terminal posttranslational processing signal (3 aa), and a propeptide (29 aa). Multiple sequence alignment results indicated that Hp1470 is an antibacterial peptide. The mature peptide Hp1470, which has a molecular mass of 1564.09 Da, was further chemically synthesized with a purity of greater than 95%. Antimicrobial assays showed that the synthesized Hp1470 exerted an inhibitory effect on Gram-positive bacteria and clinical drug-resistant strains, including PRSA and MRSA, but not Gram-negative bacteria. Hp1470 was further found to protect mice from MRSA infection, suggesting its potential application as an in vivo antimicrobial agent. Interestingly, Hp1470 only inhibited bacterial growth but did not kill bacteria, which was consistent with scanning electron microscopy results showing that Hp1470 did not lyse the cell membrane of Staphylococcus aureus. Our work provides a new direction for developing antibacterial agents with different modes of action from natural scorpion venoms.

Toxicity Effects of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) on Two Green Microalgae Species.

Amongst per- and polyfluoroalkyl substances (PFAS) compounds, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have a high persistence in physicochemical and biological degradation; therefore, the accumulation of PFOS and PFOA can negatively affect aquatic organisms and human health. In this study, two microalgae species (Chlorella vulgaris and Scenedesmus obliquus) were exposed to different concentrations of a PFOS and PFOA mixture (0 to 10 mg L-1). With increases in the contact time (days) and the PFAS concentration (mg L-1) from 1 to 7, and 0.5 to 10, respectively, the cell viability, total chlorophyll content, and protein content decreased, and the decrease in these parameters was significantly greater in Scenedesmus obliquus. As another step in the study, the response surface methodology (RSM) was used to optimize the toxicity effects of PFAS on microalgae in a logical way, as demonstrated by the high R2 (>0.9). In another stage, a molecular docking study was performed to monitor the interaction of PFOS and PFOA with the microalgae, considering hydrolysis and the enzymes involved in oxidation-reduction reactions using individual enzymes. The analysis was conducted on carboxypeptidase in Chlorella vulgaris and on c-terminal processing protease and oxidized cytochrome c6 in Scenedesmus obliquus. For the enzyme activity, the affinity and dimensions of ligands-binding sites and ligand-binding energy were estimated in each case.

Lamin A to Z in normal aging.

Almost since the discovery that mutations in the LMNA gene, encoding the nuclear structure components lamin A and C, lead to Hutchinson-Gilford progeria syndrome, people have speculated that lamins may have a role in normal aging. The most common HPGS mutation creates a splice variant of lamin A, progerin, which promotes accelerated aging pathology. While some evidence exists that progerin accumulates with normal aging, an increasing body of work indicates that prelamin A, a precursor of lamin A prior to C-terminal proteolytic processing, accumulates with age and may be a driver of normal aging. Prelamin A shares properties with progerin and is also linked to a rare progeroid disease, restrictive dermopathy. Here, we describe mechanisms underlying changes in prelamin A with aging and lay out the case that this unprocessed protein impacts normative aging. This is important since intervention strategies can be developed to modify this pathway as a means to extend healthspan and lifespan.

C-Terminal Lysine Processing of IgG in Human Suction Blister Fluid: Implications for Subcutaneous Administration.

C-terminal lysine (CTK) is often classified as a potential critical quality attribute for therapeutic antibodies being developed for subcutaneous (SC) administration because of its potential to impact antibody SC bioavailability/pharmacokinetics (PK). This classification both inflates development costs and increases comparability risks for SC administration of antibodies. However, prior risk assessments of CTK have not fully considered biotransformation of CTK in the SC space, which may play an important role given that circulating carboxypeptidases in humans rapidly process CTK on intravenously administered antibodies. Here, CTK biotransformation in biofluid derived from human SC space was investigated. The representative fluid from the human SC space was sampled from 10 healthy human subjects using the suction blister method. Glycosylated antibody containing high levels of CTK (expressed using a carboxypeptidase D CRISPR/Cas9 knockout CHO cell line) was incubated in the collected suction blister fluids (SBFs), recovered using cognate antigen pulldowns, and characterized for remaining CTK levels using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analysis. CTK processing (i.e., carboxypeptidase activity) was evident in all SBF and exhibited first-order kinetics with rate constants of 2.18 ± 0.57 d-1 (at 37 °C). PK simulations that integrated CTK processing pathways and their associated rate constants were subsequently performed using a range of clinically observed PK parameters for therapeutic antibodies, including atezolizumab- and pertuzumab-specific parameters. The impact of CTK content (even up to 100%) on SC PK outcomes such as bioavailability and Ctrough were modest (<14%) for all combinations of PK parameters tested in the sensitivity analysis. This study forms the cornerstone data package for derisking CTK as a PK liability for antibody SC programs and highlights the usefulness of fully considering biotransformation during product quality criticality assessments.

Comparison of NF-\u03baB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

The C-terminal cleavage of angiotensin II and III is mediated by prolyl carboxypeptidase in human umbilical vein and aortic endothelial cells

The renin-angiotensin system, with the octapeptide angiotensin II as key player, is important in the renal, cardiac and vascular physiology. Prolyl carboxypeptidase (PRCP), prolyl endopeptidase (PREP) and angiotensin converting enzyme 2 (ACE2) are reported to be involved in the conversion of angiotensin II to angiotensin (1–7). Previous investigations showed that the processing of angiotensin II is cell- and species-specific and little is known about its conversion in human endothelial cells. Therefore, we aimed to investigate the C-terminal processing of angiotensin II and III in comparison to the processing of des-Arg9-bradykinin in human endothelial cells. To this end, human umbilical vein and aortic endothelial cells (HUVEC and HAoEC) were incubated with the peptides for different time periods. Mass spectrometry analysis was performed on the supernatants to check for cleavage products. Contribution of PRCP, ACE2 and PREP to the peptide cleavage was evaluated by use of the selective inhibitors compound 8o, DX600 and KYP-2047. The use of these selective inhibitors revealed that the C-terminal cleavage of angiotensin II and III was PRCP-dependent in HUVEC and HAoEC. In contrast, the C-terminal cleavage of des-Arg9-bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. With this study, we contribute to a better understanding of the processing of peptides involved in the alternative renin-angiotensin system. We conclude that PRCP is the main enzyme for the C-terminal processing of angiotensin peptides in human umbilical vein and aortic endothelial cells. For the first time the contribution of PRCP was investigated by use of a selective PRCP-inhibitor.

Molecular Cloning and Functional Identification of the Antimicrobial Peptide Gene Ctri9594 from the Venom of the Scorpion Chaerilus tricostatus

Scorpion venom is a mixture of bioactive peptides, among which neurotoxins and antimicrobial peptides serve especially vital functions. Scorpion venom peptides in Buthidae species have been well described, but toxic peptides from non-Buthidae species have been under-investigated. Here, an antimicrobial peptide gene, Ctri9594, was cloned and functionally identified from the venom of the scorpion Chaerilus tricostatus. The precursor nucleotide sequence of Ctri9594 is 199 nt in length, including a 43 nt 5′ UTR, 115 nt 3′ UTR and 210 nt ORF. The ORF encodes 69 amino acid residues, containing a 21 aa signal peptide, 14 aa mature peptide, 3 aa C-terminal posttranslational processing signal and 31 aa propeptide. Multiple sequence alignment and evolutionary analyses show that Ctri9594 is an antimicrobial peptide in scorpion venom. The mature peptide of Ctri9594 was chemically synthesized with a purity greater than 95% and a molecular mass of 1484.4 Da. Minimum inhibitory concentrations (MICs) indicate that the synthesized mature peptide of Ctri9594 has inhibitory activity against Gram-positive bacteria (Bacillus thuringensis, Bacillus subtilis, Staphylococcus aureus and Micrococcus luteus) but not Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) or a fungus (Candida albicans). The antimicrobial mechanism of Ctri9594 is inferred to be related to its amphiphilic α-helix structure.

NetCleave: an open-source algorithm for predicting C-terminal antigen processing for MHC-I and MHC-II

Antigens presented on the cell surface have been subjected to multiple biological processes. Among them, C-terminal antigen processing constitutes one of the main bottlenecks of the peptide presentation pathways, as it delimits the peptidome that will be subjected downstream. Here, we present NetCleave, an open-source and retrainable algorithm for the prediction of the C-terminal antigen processing for both MHC-I and MHC-II pathways. NetCleave architecture consists of a neural network trained on 46 different physicochemical descriptors of the cleavage site amino acids. Our results demonstrate that prediction of C-terminal antigen processing achieves high accuracy on MHC-I (AUC of 0.91), while it remains challenging for MHC-II (AUC of 0.66). Moreover, we evaluated the performance of NetCleave and other prediction tools for the evaluation of four independent immunogenicity datasets (H2-Db, H2-Kb, HLA-A*02:01 and HLA-B:07:02). Overall, we demonstrate that NetCleave stands out as one of the best algorithms for the prediction of C-terminal processing, and we provide one of the first evidence that C-terminal processing predictions may help in the discovery of immunogenic peptides.

Extracellular proteolytic activation of Pseudomonas aeruginosa aminopeptidase (PaAP) and insight into the role of its non-catalytic N-terminal domain.

Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.

Loss of mature D1 leads to compromised CP43 assembly in Arabidopsis thaliana

BackgroundPhotosystem II (PSII) is a highly conserved integral-membrane multi-subunit pigment-protein complex. The proteins, pigments, lipids, and ions in PSII need to be assembled precisely to ensure a proper PSII biogenesis. D1 is the main subunit of PSII core reaction center (RC), and is usually synthesized as a precursor D1. D1 maturation by the C-terminal processing protease CtpA is essential for PSII assembly. However, the detailed mechanism about how D1 maturation affects PSII assembly is not clearly elucidated so far. In this study, Arabidopsis thaliana CtpA mutant (atctpa: SALK_056011), which lacks the D1 mature process, was used to investigate the function of this process on PSII assembly in more details.ResultsWithout the C-terminal processing of precursor D1, PSII assembly, including PSII monomer, dimer, especially PSII supercomplexes (PSII SCs), was largely compromised as reported previously. Western blotting following the BN-2D-SDS PAGE revealed that although the assembly of PSII core proteins D2, CP43 and CP47 was affected by the loss of D1 mature process, the incorporation of CP43 was affected the most, indicated by its most reduced assembly efficiency into PSII SCs. Furthermore, the slower growth of yeast cells which were co-transformed with pD1 and CP43, when compared with the ones co-transformed with mature D1 and CP43, approved the existence of D1 C-terminal tail hindered the interaction efficiency between D1 and CP43, indicating the physiological importance of D1 mature process on the PSII assembly and the healthy growth of the organisms.ConclusionsThe knockout Arabidopsis atctpa mutant is a good material to study the unexpected link between D1 maturation and PSII SCs assembly. The loss of D1 maturation mainly affects the incorporation of PSII core protein CP43, an inner antenna binding protein, which functions in the association of LHCII complexes to PSII dimers during the formation of PSII SCs. Our findings here provide detailed supports of the role of D1 maturation during PSII SCs assembly in higher plants.

Lacritin proteoforms prevent tear film collapse and maintain epithelial homeostasis

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Properties and Biotechnological Application of Mutant Derivatives of the Mini-Intein PRP8 from Penicillium chrysogenum with Improved Control of C-Terminal Processing

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing are characterized. The studied variants can serve as the basis for the self-removal of polypeptide tags that can carry an affine label and optimize the process of the obtainment of target proteins and peptides in E. coli cells. They make it possible to synthesize target molecules composed of soluble and insoluble hybrid proteins (fusions), to conduct their affine purification and autocatalytic processing, and to obtain mature target products. The presented variants have a number of features as compared to the known prototypes. In particular, the mutant mini-intein Int4bPRO, which contains the L93P mutation, has temperature-dependent properties. It is able to produce target molecules composed of soluble fusions at a cultivation temperature below 30°C; however, it directs most synthesized fusions into insoluble intracellular aggregates after an increase in temperature to 37°C. The transition of Int4bPRO to an insoluble form is accompanied by the complete inactivation of C-terminal processing. Further application of the standard protein denaturation–renaturation procedures enables efficient reactivation of Int4bPRO and the processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, which contain the point mutation T62N or a combination of D144N and L146T mutations, respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows optimization of the biosynthesis of biologically active target proteins and peptides composed of soluble fusions that are suitable for affine purification and subsequent intein-dependent processing without the use of protein denaturation–renaturation procedures.

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability.

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability.

Processing and Formation of Bioactive CLE40 Peptide Are Controlled by Posttranslational Proline Hydroxylation.

Small posttranslationally modified signaling peptides are proteolytically derived from larger precursor proteins and subject to several additional steps of modification, including Pro hydroxylation, Hyp glycosylation, and/or Tyr sulfation. The processing proteases and the relevance of posttranslational modifications for peptide biogenesis and activity are largely unknown. In this study these questions were addressed for the Clavata3/Endosperm Surrounding Region (CLE) peptide CLE40, a peptide regulator of stem cell differentiation in the Arabidopsis (Arabidopsis thaliana) root meristem. We identify three subtilases (SBT1.4, SBT1.7, and SBT4.13) that cleave the CLE40 precursor redundantly at two sites. C-terminal processing releases the mature peptide from its precursor and is thus required for signal biogenesis. SBT-mediated cleavage at a second site within the mature peptide attenuates the signal. The second cleavage is prevented by Pro hydroxylation, resulting in the formation of mature and bioactive CLE40 in planta. Our data reveal a role for posttranslational modification by Pro hydroxylation in the regulation of CLE40 formation and activity.

The pH-sensing Rim101 pathway positively regulates the transcriptional expression of the calcium pump gene PMR1 to affect calcium sensitivity in budding yeast

In Saccharomyces cerevisiae, the Rim101 pathway senses extracellular pH changes through a complex consisted of Rim8, Rim9 and Rim21 at the plasma membrane. Activation of this sensor complex induces a proteolytical complex composed of Rim13 and Rim20 and leads to the C-terminal processing and activation of the transcription factor Rim101. Deletion mutants for RIM8, RIM9, RIM13, RIM20, RIM21 and RIM101 causes yeast cells to be sensitive to calcium stress, but how they regulate calcium sensitivity remain unknown. Here we show that deletion mutations of these six Rim101 pathway components elevate the activation level of the calcium/calcineurin signaling and the transcriptional expression level of the vacuolar calcium pump gene PMC1, but lead to a reduction in transcriptional expression level of the ER/Golgi calcium pump gene PMR1 in yeast cells. Deletion of NRG1, encoding one of the repression targets of Rim101, rescues the transcriptional expression of PMR1 in all these mutants. Furthermore, ectopic expression of a constitutively active form of Rim101 or further deletion of NRG1 suppresses the calcium sensitivity of these six deletion mutants. Therefore, the pH-sensing Rim101 pathway positively regulates the transcriptional expression of PMR through its downstream target Nrg1 to affect the calcium sensitivity of yeast cells.

Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase.

Chloroplast proteostasis is governed by a network of peptidases. As a part of this network, we show that Arabidopsis (Arabidopsis thaliana) chloroplast glutamyl peptidase (CGEP) is a homo-oligomeric stromal Ser-type (S9D) peptidase with both exo- and endo-peptidase activity. Arabidopsis CGEP null mutant alleles (cgep) had no visible phenotype but showed strong genetic interactions with stromal CLP protease system mutants, resulting in reduced growth. Loss of CGEP upregulated the chloroplast protein chaperone machinery and 70S ribosomal proteins, but other parts of the proteostasis network were unaffected. Both comparative proteomics and mRNA-based coexpression analyses strongly suggested that the function of CGEP is at least partly involved in starch metabolism regulation. Recombinant CGEP degraded peptides and proteins smaller than ∼25 kD. CGEP specifically cleaved substrates on the C-terminal side of Glu irrespective of neighboring residues, as shown using peptide libraries incubated with recombinant CGEP and mass spectrometry. CGEP was shown to undergo autocatalytic C-terminal cleavage at E946, removing 15 residues, both in vitro and in vivo. A conserved motif (A[S/T]GGG[N/G]PE946) immediately upstream of E946 was identified in dicotyledons, but not monocotyledons. Structural modeling suggested that C-terminal processing increases the upper substrate size limit by improving catalytic cavity access. In vivo complementation with catalytically inactive CGEP-S781R or a CGEP variant with an unprocessed C-terminus in a cgep clpr2-1 background was used to demonstrate the physiological importance of both CGEP peptidase activity and its autocatalytic processing. CGEP homologs of photosynthetic and nonphotosynthetic bacteria lack the C-terminal prosequence, suggesting it is a recent functional adaptation in plants.