Propeptide Domain Research Articles

Abstract 4473: Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous ovarian cancer

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most aggressive and lethal gynecological malignancy. Surgery and chemotherapy (with platinum compounds) are the main treatments. Many patients relapse and develop chemotherapy resistance, limiting the therapeutic utility of these therapies. This persistent resistance makes HGSOC treatment difficult. Cisplatin-resistant ovarian cancer cells upregulate Matrix Metalloproteinase-3. MMP3 is an enzyme involved in active tissue remodeling and extracellular matrix protein breakdown during dynamic physiological processes. The structure of MMP3 includes the prodomain, the catalytic domain and the hemopexin domain. We showed that MMP3 protein levels are higher in cisplatin resistant as compared with cisplatin sensitive HGSOC cells. Our research team showed that small interference RNA (siRNA)-mediated MMP3 knockdown reduced cisplatin-resistant ovarian cancer cell growth and invasion. This data indicates that MMP3 may be a promising HGSOC treatment target. Targeting MMP3 with small chemical inhibitors (SCI) could represent a better option than siRNA because SCI are more stable and easier to deliver. The purpose of this project is to design SCI of MMP3 in the propeptide, catalytic, and hemopexin domains of MMP3. We used in silico methods to identify MMP3 inhibitors in these domains. The ZINC20 database of commercially compounds was used for virtual screening using Python Prescription (PyRx) software. We identified 25 ligands out of 370,000 with the greatest affinity for each MMP3 functional domains. SwissADME was then used to evaluate in silico absorption, distribution, metabolism, pharmacokinetics, drug-likeliness, and medicinal chemistry. This research lays the groundwork for HGSOC therapeutic strategies to improve treatment efficacy, especially for chemotherapy-resistant patients. Citation Format: Víctor G. Reyes-Burgos, Eliud Hernández-O'Farrill, Luis E. Vazquez Quiñones, Marienid Flores-Colón, Fatima Valiyeva, Pablo E. Vivas-Mejía. Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4473.

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

In silico molecular characterization of TGF-β gene family in Bufo bufo: genome-wide analysis

Bufo bufo is a living example of evolutionary processes due to its numerous physiological and ecological adaptations. This is the first study to genetically characterize the TGF-β gene family in B. bufo at the genome-wide level, and a total of 28 TGF-β gene family homologs are identified. Physicochemical characteristics of TGF-β homologs exhibit a basic nature except for BMP1, BMP4, BMP10, BMP15, AMH, INHA, NODAL Modulator and TGFB1. Phylogenetic analysis divided the TGF-β gene family homologs into 2 major clades along with other vertebrate species. In domain and motif composition analysis, the gene structure for all TGF-β homologs exhibited homogeneity except BMP1. We have identified the TGF-β propeptide domain together with the TGF-β in all family homologs of TGF-β superfamily. Gene structure comparisons indicated that the TGF-β gene family have arisen by gene duplications. We also identified 10 duplicated gene pairs, all of which were detected to be segmental duplications. The Ka/Ks test ratio findings for every pair of genes revealed that none of the ratios surpassed 1 except for one gene pair (INHA/BMP1), indicating that these proteins are under positive selection. Circos analysis showed that TGF-β gene family homologs are arranged in 11 dispersed clusters and all were segmentally arrayed in the genome. This study provides a molecular basis for TGF-β ligand protein functional analysis and may serve as a reference for in-depth phylogenomics and may promote the development of novel strategies. Communicated by Ramaswamy H. Sarma

Functional Characterization of the Almstn2 Gene and Its Association with Growth Traits in the Yellowfin Seabream Acanthopagrus latus (Hottuyn, 1782).

Myostatin (mstn), also known as GDF8, is a growth and differentiation factor of the transforming growth factor-β (TGF-β) superfamily and plays a key inhibitory effect in the regulation of skeletal muscle development and growth in vertebrates. In the present study, to comprehend the role of the mstn2 gene of the yellowfin seabream Acanthopagrus latus (Almstn2b), the genomic sequence of Almstn2b is 2359 bp, which encodes 360 amino acids and is composed of three exons and two introns, was obtained. Two typical regions, a TGF-β propeptide and TGF-β domain, constitute Almstn2b. The topology indicated that Almstn2 was grouped together with other Perciformes, such as the gilthead seabream Sparus aurata. Moreover, Almstn2b was mainly expressed in the brain, fins, and spleen. Furthermore, five SNPs, one in the exons and four in the introns, were identified in the Almstn2b gene. The allele and genotype frequencies of SNP-Almstn2b +1885 A/G were significantly related to the total weight, interorbital distance, stem length, tail length, caudal length, caudal height, body length, and total length (p < 0.05). The allele and genotype frequencies of SNP-Almstn2b +1888 A/G were significantly related to the weight, interorbital distance, long head behind the eyes, body height, tail length, caudal length, and body length. Additionally, the relationship between the SNP-Almstn2b +1915 A/G locus and weight and long head behind the eyes was significant (p < 0.05). Furthermore, the other two SNPs were not significantly associated with any traits. Thus, the SNPs identified in this study could be utilized as candidate SNPs for breeding and marker-assisted selection in A. latus.

High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease.

Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homologous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autoprocessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demonstrated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules. IMPORTANCE Many microorganisms secrete proteases into their environment to degrade protein substrates for their growth. The important group of these extracellular enzymes are subtilases, which are also widely used in practical applications. These subtilases are inhibited by their propeptide domain, which is degraded during the prosubtilase maturation process. Here, we showed that the propeptide of pernisine, a prion-degrading subtilase from the hyperthermophilic archaeon, strongly inhibits pernisine with extraordinarily high binding affinity. This interaction proved to be highly selective, as pernisine propeptide was rapidly degraded by mesophilic pernisine homologs. This in turn allowed rapid transactivation of propernisine by mesophilic subtilases at lower temperatures, which might simplify the procedures for preparation of active pernisine for commercial use. The results reported in this study suggest that the hyperthermophilic subtilase propeptide evolved to function as tight and selective regulator of maturation of the associated prosubtilase to prevent its premature activation under high temperatures.

Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2.

Nerve growthfactor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR , and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+ , known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis.

A Potential Negative Regulatory Function of Myostatin in the Growth of the Pacific Abalone, Haliotis discus hannai.

Myostatin, also known as GDF8, is a member of the transforming growth factor-β (TGF-β) superfamily. In vertebrates, myostatin negatively regulates the growth of skeletal muscle. In invertebrates, it has been reported to be closely related to animal growth. However, knowledge concerning the molecular mechanisms involved in the myostatin regulation of molluscan growth is limited. In this study, we found that the hdh-myostatin open reading frame (ORF) comprised 1470 base pairs that encoded 489 amino acids and contained structural characteristics typical of the TGF-β superfamily, including a C-terminal signal peptide, a propeptide domain, and TGF-β region. Gene expression analysis revealed that hdh-myostatin mRNA was widely expressed at different levels in all of the examined tissues of Haliotis discus hannai. Nine single nucleotide polymorphisms (SNPs) were associated with the growth traits. RNA interference (RNAi) against hdh-myostatin mRNA significantly downregulated hdh-myostatin at days 1, 15, and 30 post injection, and the pattern was correlated with downregulation of the genes TGF-β receptor type-I (hdh-TβR I), activin receptor type-IIB (hdh-ActR IIB), and mothers against decapentaplegic 3 (hdh-Smad3). After one month of the RNAi experiment, the shell lengths and total weights increased in the abalone, Haliotis discus hannai. The results of qRT-PCR showed that the hdh-myostatin mRNA level was higher in the slow-growing group than in the fast-growing group. These results suggest that hdh-myostatin is involved in the regulation of growth, and that these SNPs would be informative for further studies on selective breeding in abalone.

Collagen a1(XI) structure prediction by Alphafold 2.

Collagen α1(XI) is a minor fibrillar collagen involved in the critical regulation of collagen fibrils such as nucleation, assembly, and regulation of fibril diameter. The amino propeptide domain of the collagen α1(XI) is retained on the surface of the collagen fibril for an extended period of time and may play a crucial role in the interaction with extracellular matrix glycosaminoglycans and other proteins during the process of fibrillogenesis. Understanding the mechanism of action of this protein will ultimately help us understand the organization and assembly of the extracellular matrix that underlies the structural integrity of connective tissues.

In Silico Study and Effects of BDMC33 on TNBS-Induced BMP Gene Expressions in Zebrafish Gut Inflammation-Associated Arthritis

The bone morphogenic protein (BMP) family is a member of the TGF-beta superfamily and plays a crucial role during the onset of gut inflammation and arthritis diseases. Recent studies have reported a connection with the gut-joint axis; however, the genetic players are still less explored. Meanwhile, BDMC33 is a newly synthesized anti-inflammatory drug candidate. Therefore, in our present study, we analysed the genome-wide features of the BMP family as well as the role of BMP members in gut-associated arthritis in an inflammatory state and the ability of BDMC33 to attenuate this inflammation. Firstly, genome-wide analyses were performed on the BMP family in the zebrafish genome, employing several in silico techniques. Afterwards, the effects of curcumin analogues on BMP gene expression in zebrafish larvae induced with TNBS (0.78 mg/mL) were determined using real time-qPCR. A total of 38 identified BMP proteins were revealed to be clustered in five major clades and contain TGF beta and TGF beta pro peptide domains. Furthermore, BDMC33 suppressed the expression of four selected BMP genes in the TNBS-induced larvae, where the highest gene suppression was in the BMP2a gene (an eight-fold decrement), followed by BMP7b (four-fold decrement), BMP4 (four-fold decrement), and BMP6 (three-fold decrement). Therefore, this study reveals the role of BMPs in gut-associated arthritis and proves the ability of BDMC33 to act as a potential anti-inflammatory drug for suppressing TNBS-induced BMP genes in zebrafish larvae.

Comprehensive Characterization of Missense Variation in Coagulation Factor IX Reveals Features Necessary for Secretion and γ-Carboxylation

Multiplexed Assays of Variant Effect (MAVEs) are a powerful tool for assessing the effects of variation in proteins to understand sequence-function relationships, inform biological structures, and infer pathogenicity in clinical settings. We wished to use MAVEs to study the functional impact of all possible missense variants on coagulation factor IX (FIX). Variation in the F9 gene can cause decreased levels of functional FIX, leading to the bleeding disorder hemophilia B. However, genes that can be studied with MAVEs were limited to those that encode intracellular or membrane-bound proteins. This is because each variant protein must be physically connected to the genome of the cell of origin to allow functional protein selection and subsequent DNA sequencing to measure individual variant effects. However, FIX is a secreted plasma protein that does not remain physically associated with the cell that expresses it, making it incompatible with current MAVE methods. We hypothesized that cell surface display, as has been used in yeast and phage, could be adapted to establish the connection between FIX protein and F9 DNA variation needed for MAVEs in mammalian cells. Thus, we developed a FIX mammalian cell surface display system in HEK-293 cells by fusing FIX protein to a single pass transmembrane domain with a C-terminal linker. We measured FIX expression in our mammalian cell-surface display system using flow cytometry and multiple monoclonal, fluorescent anti-FIX antibodies to different FIX domains. We introduced a library of nearly all ~10,000 FIX missense variants into this system to profile each variant's effect on FIX function. Antibody-stained cells were then sorted into quartile bins by FACS. Variants in each bin were then deeply sequenced and scored based on their frequency within each bin. Scores were then used to assign functional significance to each FIX variant. To detect variants that impact FIX secretion, we used both antibodies to both FIX heavy chain and a strep II epitope engineered into the C-terminal linker. With the anti-FIX heavy chain antibody, we found that approximately 36.2% of variants were lowly secreted and that secretion scores alone correlated well with FIX antigen data reported in the Factor IX (F9) Gene Variant Database. We further found that proline and cysteine substitutions had strong negative effects on FIX expression. The anti-strep II antibody yielded similar results, except that cysteine substitution was generally better-tolerated. We concluded that the FIX heavy chain antibody binding is particularly sensitive to loss of function due to cysteine substitutions. FIX post-translational modification, particularly γ-carboxylation of the GLA domain, is critical for normal FIX function. A γ-carboxylation-sensitive anti-FIX monoclonal antibody can detect properly γ-carboxylated FIX. In our system, binding of this γ-carboxylation-sensitive antibody was lost when cells were grown in the presence of warfarin, indicating that our displayed FIX is γ-carboxylated. We used this carboxylation-sensitive antibody to detect FIX variants associated with reduced FIX carboxylation-sensitive antibody binding relative to secretion. As expected, the majority of variants with disproportionately low γ-carboxylation were located within the propeptide and GLA domains. Thus, we developed a novel membrane-tethered mammalian display system to study the functional impact of all possible missense variation in the secreted coagulation protein FIX. Our results provide direct functional evidence for loss of function in a high proportion (39.6%) of all possible FIX missense variants, either by decreasing secretion or by loss of γ-carboxylation.

A propeptide-based biosensor for the selective detection of Vibrio cholerae using an environment-sensitive fluorophore

Proteases are attractive targets for infectious disease diagnostics. Peptide-based sensors that are cleaved by pathogen proteases can provide a rapid readout of infection. However, identifying peptide substrates specific to a targeted pathogen is a significant challenge. Here, we demonstrate that a structured propeptide domain from a bacterial protease can be repurposed as a protease-activated biosensor of the cholera pathogen Vibrio cholerae. We found that the peptidase inhibitor I9 domain of the secreted V.cholerae protease IvaP is rapidly degraded by V.cholerae, but not by other intestinal bacteria. By conjugating the I9 domain to an environment-sensitive fluorophore, we developed a fluorescent probe that enables the species-specific detection of V.cholerae in mixed bacterial cultures without nonspecific cleavage by other bacteria or intestinal cells. Our findings demonstrate that the IvaP propeptide is sufficient to impart selectivity to a cleavage-based V.cholerae biosensor, suggesting I9 domains could potentially be harnessed for diagnostic applications.

Characterization of a novel cathepsin D and its immunity response in noble scallop Chlamys nobilis with different total carotenoids content

Cathepsin D is a soluble lysosomal aspartic proteinase of the pepsin superfamily that plays a crucial role in immune response. In this study, a novel cathepsin D of noble scallop Chlamys nobilis (named as CnCD) was cloned and its immune response to three different immunostimulants, including polyinosinic polycytidylic acid, lipo-polysaccharide and Vibrio parahaemolyticus, was investigated in golden scallops and brown scallops of C. nobilis with different carotenoids content. The full-length cDNA of CnCD is 1844 bp, containing a 63-bp 5′ terminal untranslated region (5′-UTR), a 1212-bp ORF encoding 403 amino acids and a 569-bp 3′-UTR. The predicted protein sequence of CnCD is composed of a signal peptide, a propeptide domain and a mature domain, and has a conserved bilobal structure. Phylogenetic analysis showed the CnCD was clustered into invertebrate group, and especially close to mollusc. Tissue distribution analysis of CnCD showed it was widely distributed in all tested tissues with higher levels in intestine, haemocytes and hepatopancreas. After immunostimulants challenged, CnCD transcripts were significantly up-regulated compared with the control group, indicating the CnCD plays important roles in immune response. Furthermore, the expression levels of CnCD in golden scallops were significantly higher than that of brown ones at most time points, suggesting carotenoids in golden scallops are important in promoting the immune response against pathogens. These results suggested that CnCD plays important roles in the immune defence against pathogens and the carotenoids can enhance the immune ability by up-regulating immune-related genes in noble scallop.

Exploring mutable conserved sites and fatal non-conserved sites by random mutation of esterase from Sulfolobus tokodaii and subtilisin from Thermococcus kodakarensis

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites. In this study, we explored mutable conserved sites and immutable non-conserved sites by testing random mutations of two thermostable proteins, an esterase from Sulfolobus tokodaii (Sto-Est) and a subtilisin from Thermococcus kodakarensis (Tko-Sub). The subtilisin domain of Tko-Sub needs Ca2+ ions and the propeptide domain for stability, folding and maturation. The results from the two proteins showed that about one-third of the mutable sites were detected in conserved sites and some non-conserved sites lost enzymatic activity at high temperatures due to mutation. Of the conserved sites in Sto-Est, the sites on the loop, on the surface, and far from the active site are more resistant to mutation. In Tko-Sub, the sites flanking Ca2+-binding sites and propeptide were undesirable for mutation. The results presented here serve as an index for selecting mutation sites and contribute to the expansion of available sequence range by introducing mutations at conserved sites.

Identification and expression analysis of Cathepsin B-like protease 2 genes in tomato at abiotic stresses especially at High temperature

Cathepsin B-like protease 2 (CathB2) genes have been shown to participate in programmed cell death (PCD) that is a crucial component of development and defense mechanisms in plants at biotic and abiotic stresses. However, little is known regarding CathB2 genes in tomato. In this study, we reported the analysis of two SlCathB2 genes named SlCathB2−1 and SlCathB2−2. Promoter analysis found some elements related to abiotic stress-responses in two SlCathB2 genes. Domain analysis revealed that SlCathB2 encoding proteins characterized by signal peptide, propeptide, and papain family cysteine protease domain. The phylogenetic analysis revealed their close evolutionary relationship with the CathB2 of Solanum tuberosum. Expression analysis showed that SlCathB2 differentially expressed in various tissues and SlCathB2 positively responded to high temperature, drought, salt, ABA and SA treatments with higher expression upon high-temperature stress (HS) in the cultivated tomato line ‘LA1698′ (Solanum lycopersicum) and the currant tomato line ‘LA2093′ (Solanum pimpinellifolium). Then, the expression of SlCathB2 in the leaves of tomato genotypes with different thermotolerance under moderately elevated temperature (MET, 33 °C/33 °C) and acutely elevated temperature (AET, 40 °C/40 °C) stresses were further analyzed. The results showed that SlCathB2 genes were most triggered in heat-sensitive tomato genotypes (S1 and S2) as compared to heat-tolerant genotypes (T1 and T2) and were not significantly induced in the moderate genotypes (M1 and M2) under MET and AET stresses, suggesting that they were negatively regulated by HS. Furthermore, SlCathB2 genes showed similar expression patterns in tomato genotypes with same behavior for thermotolerance under MET and AET stresses, while exhibited higher expression level under AET stress, indicating that more thermosensors and multiple HSR (high-temperature stress- responsive) pathways were activated under AET stress leading to the higher expression level of a set of HSR genes including SlCathB2−1 and SlCathB2−2 genes. This study lays a foundation for functional studies of SlCathB2 in tomato.

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem

Recent studies have reported that aphids facilitate their colonization of host plants by secreting salivary proteins into host tissues during their initial probing and feeding. Some of these salivary proteins elicit plant defenses, but the molecular and biochemical mechanisms that underlie the activation of phloem-localized resistance remain poorly understood. The aphid Myzus persicae, which is a generalized phloem-sucking pest, encompasses a number of lineages that are associated with and adapted to specific host plant species. The current study found that a cysteine protease Cathepsin B3 (CathB3), and the associated gene CathB3, was upregulated in the salivary glands and saliva of aphids from a non-tobacco-adapted (NTA) aphid lineage, when compared to those of a tobacco-adapted lineage. Furthermore, the knockdown of CathB3 improved the performance of NTA lineages on tobacco, and the propeptide domain of CathB3 was found to bind to tobacco cytoplasmic kinase ENHANCED DISEASE RESISTANCE 1-like (EDR1-like), which triggers the accumulation of reactive oxygen species in tobacco phloem, thereby suppressing both phloem feeding and colonization by NTA lineages. These findings reveal a novel function for a cathepsin-type protease in aphid saliva that elicits effective host plant defenses and warranted the theory of host specialization for generalist aphids.

Identification of Bacterial Protein Interaction Partners Points to New Intracellular Functions of Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA’s interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.

Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions.

Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Thermal stability of human matrix metalloproteinases

Matrix metalloproteinases (MMP) are key players in the remodelling of the extracellular matrix under physiological and pathological conditions. Thermodynamic parameters of human recombinant metalloproteinases of the active (rMMP2, 3, 7, 8 and 9) and latent (rPro-MMP2, 3 and 9) forms were obtained by differential scanning calorimetry (DSC). Temperature by itself does not result in autocatalysis of recombinant MMP. The transitions observed by DSC correspond to structural domains of the monomeric protein. In this study, we show the domain organization of these proteins, where the thermal transition (Tm) of the main component is observed at 71.3 °C (ProMMP-2); 74.8 °C (ProMMP-8); 80.0 °C (ProMMP-3); 92.6 °C (ProMMP-9) and 98.3 °C (ProMMP-7). For MMP-3, this main Tm is related to the catalytic domain (CD). The isolated recombinant CD of MMP-3 unfolds as a single transition at Tm 83.4 °C, matching the more stable domain observed in the full-length active form of rMMP-3. The denaturation profile of rProMMP-3 shows the main transition at Tm 80 °C, a less stable domain before the propeptide domain (PD) cleavage. Our results indicate that the structural stability of MMP and particularly their CD are not substantially altered after cleavage of the PD. We propose that the thermodynamic parameters obtained by DSC are relevant for the functional study of MMP, particularly to reveal their contribution in complex biological samples in health and disease.

Matrix Metalloproteinases in Invertebrates

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.