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Telomeres are protein–DNA complexes that protect chromosome ends from illicit ligation and resection. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA to counter telomere shortening. Human telomeres are composed of complexes between telomeric DNA and a six-protein complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a network of constitutive and context-specific interactions. The shelterin protein–DNA structures reveal the basis for both the high affinity and DNA sequence specificity of these interactions, and explain how shelterin efficiently protects chromosome ends from genome instability. Several protein–protein interactions, many provided by the shelterin component TIN2, are critical for upholding the end-protection function of shelterin. A survey of these protein–protein interfaces within shelterin reveals a series of “domain–peptide” interactions that allow for efficient binding and adaptability towards new functions. While the modular nature of shelterin has facilitated its part-by-part structural characterization, the interdependence of subunits within telomerase has made its structural solution more challenging. However, the exploitation of several homologs in combination with recent advancements in cryo-EM capabilities has led to an exponential increase in our knowledge of the structural biology underlying telomerase function. Telomerase homologs from a wide range of eukaryotes show a typical retroviral reverse transcriptase-like protein core reinforced with elements that deliver telomerase-specific functions including recruitment to telomeres and high telomere-repeat addition processivity. In addition to providing the template for reverse transcription, the RNA component of telomerase provides a scaffold for the catalytic and accessory protein subunits, defines the limits of the telomeric repeat sequence, and plays a critical role in RNP assembly, stability, and trafficking. While a high-resolution definition of the human telomerase structure is only beginning to emerge, the quick pace of technical progress forecasts imminent breakthroughs in this area. Here, we review the structural biology surrounding telomeres and telomerase to provide a molecular description of mammalian chromosome end protection and end replication.

Plant protease inhibitors (PPI) play a significant role against microbes, insects, and, to a considerable extent, human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of . The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor was predominantly composed of α-helical content. The kinetics experiments demonstrated a noncompetitive mode of inhibition towards serine protease when casein was used as the enzyme substrate. The inhibitor formed a stable complex with serine protease, having a likely 1:1 stoichiometry, as inferred from ITC, and the dissociation constant was examined to be K = 1.9 × 10 M with a Gibbs free energy of ΔG = -8.079 (kcal/mol). The inhibitor exhibits stable protease inhibition up to 90 °C. Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence that it may have a role in the treatment of viral infections.

Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model mechanisms can result in decade long discussions about the correct interpretation of data and the true theory behind it. However, often such opposing views turn out to be special cases of a more comprehensive and superior concept. Molecular dynamics (MD) and the more advanced molecular mechanical and quantum mechanical approach (QM/MM) provide a relatively consistent framework to treat enzymatic mechanisms, in particular, the activity of proteolytic enzymes. In line with this, computational chemistry based on experimental structures came up with studies on all major protease classes in recent years; examples of aspartic, metallo-, cysteine, serine, and threonine protease mechanisms are well founded on corresponding standards. In addition, experimental evidence from enzyme kinetics, structural research, and various other methods supports the described calculated mechanisms. One step beyond is the application of this information to the design of new and powerful inhibitors of disease-related enzymes, such as the HIV protease. In this overview, a few examples demonstrate the high potential of the QM/MM approach for sophisticated pharmaceutical compound design and supporting functions in the analysis of biomolecular structures.

Commensal bacteria are known to inhibit pathogen colonization; however, complex host–microbe and microbe–microbe interactions have made it difficult to gain a detailed understanding of the mechanisms involved in the inhibition of colonization 1 . Here we show that the serine protease Esp 2 , 3 secreted by a subset of Staphylococcus epidermidis , a commensal bacterium, inhibits biofilm formation and nasal colonization by Staphylococcus aureus , a human pathogen 4 . Epidemiological studies have demonstrated that the presence of Esp-secreting S. epidermidis in the nasal cavities of human volunteers correlates with the absence of S. aureus . Purified Esp inhibits biofilm formation and destroys pre-existing S. aureus biofilms. Furthermore, Esp enhances the susceptibility of S. aureus in biofilms to immune system components. In vivo studies have shown that Esp-secreting S. epidermidis eliminates S. aureus nasal colonization. These findings indicate that Esp hinders S. aureus colonization in vivo through a novel mechanism of bacterial interference, which could lead to the development of novel therapeutics to prevent S. aureus colonization and infection.

Key Points Several lines of evidence have implicated the high temperature requirement A (HTRA) family of homooligomeric Ser proteases in protein quality control. HTRA proteases are implicated in bacterial virulence and stress response, photosynthesis in plants, and proliferation, migration and cell fate in mammals. HTRA proteases share common principles of activation with classic Ser proteases such as trypsin, chymotrypsin and elastase. However, their unique architecture, including carboxy-terminal PDZ (postsynaptic density of 95 kDa, Discs large and zonula occludens 1) domains, is responsible for a remarkable structural and functional plasticity that allows cells to rapidly respond to the presence of misfolded or mislocalized polypeptides. The activity of HTRA proteases is tightly regulated by their switching on and off by peptides that bind to the PDZ domains. Activation is usually reversible and can involve a change in oligomeric state. The PDZ domains of HTRA proteases are involved in a great variety of functions, including allosteric activation, cooperativity, processivity, activation by oligomerization, cellular localization (including lipid binding) and sensing of protein-folding stress. HTRA proteases perform a variety of protein quality control functions that are of key importance to cell fate. This Review discusses the emerging physiological implications and unique architectural and mechanistic features of bacterial, plant and mammalian HTRAs. Controlled proteolysis underlies a vast diversity of protective and regulatory processes that are of key importance to cell fate. The unique molecular architecture of the widely conserved high temperature requirement A (HTRA) proteases has evolved to mediate critical aspects of ATP-independent protein quality control. The simple combination of a classic Ser protease domain and a carboxy-terminal peptide-binding domain produces cellular factors of remarkable structural and functional plasticity that allow cells to rapidly respond to the presence of misfolded or mislocalized polypeptides.

Subtilisin-like proteins are serine proteases that use two types of catalytic triads: Ser-His-Asp and Ser-Glu-Asp. Here, we investigate the two known families of subtilisin-like proteins, the subtilases (Ser-His-Asp triad) and the serine-carboxyl proteinases (Ser-Glu-Asp triad), and describe the local structural arrangements (cores) that govern the catalytic residues in these proteins. We show the separation of the cores into conserved structural zones, which can be repeatedly found in different structures, and compare the structural cores in subtilisin-like proteins with those in trypsin-like serine proteases and alpha/beta-hydrolases.

Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs’ contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40-60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Sperm proteases are involved in several gamete interaction events leading to fertilization. This report presents a detailed analysis of the expression and localization of serine protease PRSS38 in human and in mouse spermatozoa and its involvement in fertilization-related events, using bioinformatics, cellular, biochemical, molecular, and functional approaches. Bioinformatics analyses included genomics and data analysis, prediction of protein subcellular localization and post-translational modifications, Self-Organizing Maps (SOMs) unsupervised training with other serine proteases, protein modeling (AlphaFold), and genetic variant analysis. For cellular, biochemical, and functional studies, human semen samples were obtained from healthy normozoospermic volunteers, and epididymal sperm were collected from adult Balb-c/C57 mice. PRSS38 presence was detected in human and mouse sperm protein extracts by Western immunoblotting. Sperm PRSS38 subcellular localization was determined by fluorescence immunocytochemistry. Human sperm-oocyte interaction events were assessed by means of the mouse Cumulus Penetration Assay (CPA) using mouse COCs, the Human Hemizona Assay (HZA), and the ZP-free hamster egg Sperm Penetration Assay (SPA). Mouse sperm-oocyte interactions were evaluated by means of in vitro fertilization (IVF) with COCs and denuded oocytes. PRSS38 is proposed to be a GPI-anchored serine protease (active site: His-100, Asp-150, and Ser-245) based on bioinformatics analyses. Using commercial antibodies, protein forms of the expected Mr (human: 31 kDa; mouse: 32 and 24 kDa) were specifically immunodetected in protein sperm extracts. Immunocytochemical analysis revealed a specific PRSS38 signal in the human sperm acrosomal region, equatorial segment, and flagellum. Mouse sperm PRSS38 was immunolocalized in the equatorial segment and hook. Human sperm preincubation with specific antibodies resulted in inhibition ( < 0.05) of CPA, HZA, and SPA. Mouse sperm preincubation with PRSS38 antibodies impaired ( < 0.05) homologous IVF using COCs and denuded oocytes. Genetic variants affecting residues involved in the GPI anchor and the catalytic triad were found in individuals from the general population whose PRSS38 protease function could be altered. This study provides, for the first time, an integrated analysis of PRSS38 in human and mouse sperm, contributing to our understanding of mammalian fertilization and male infertility.