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Journal of Proteomics Oct 2023Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate...
Here we introduce hyperthermoacidic archaeal proteases (HTA-Proteases©) isolated from organisms that thrive in nearly boiling acidic volcanic springs and investigate their use for bottom-up proteomic experiments. We find that HTA-Proteases have novel cleavage specificities, show no autolysis, function in dilute formic acid, and store at ambient temperature for years. HTA-Proteases function optimally at 70-90 °C and pH of 2-4 with rapid digestion kinetics. The extreme HTA-Protease reaction conditions actively denature sample proteins, obviate the use of chaotropes, are largely independent of reduction and alkylation, and allow for a one-step/five-minute sample preparation protocol without sample manipulation, dilution, or additional cleanup. We find that brief one-step HTA-Protease protocols significantly increase proteome and protein sequence coverage with datasets orthogonal to trypsin. Importantly, HTA-Protease digests markedly increase coverage and identifications for ribonucleoproteins, histones, and mitochondrial membrane proteins as compared to tryptic digests alone. In addition to increased coverage in these classes, HTA-Proteases and simplified one-step protocols are expected to reduce technical variability and advance the fields of clinical and high-throughput proteomics. This work reveals significant utility of heretofore unavailable HTA-Proteases for proteomic workflows. We discuss some of the potential for these remarkable enzymes to empower new proteomics methods, approaches, and biological insights. SIGNIFICANCE: Here we introduce new capabilities for bottom-up proteomics applications with hyperthermoacidic archaeal proteases (HTA-Proteases©). HTA-Proteases have novel cleavage specificity, require no chaotropes, and allow simple one-step/five-minute sample preparations that promise to reduce variability between samples and laboratories. HTA-Proteases generate unique sets of observable peptides that are non-overlapping with tryptic peptides and significantly increase sequence coverage and available peptide targets relative to trypsin alone. HTA-Proteases show some bias for the detection and coverage of nucleic acid-binding proteins and membrane proteins relative to trypsin. These new ultra-stable enzymes function optimally in nearly boiling acidic conditions, show no autolysis, and do not require aliquoting as they are stable for years at ambient temperatures. Used independently or in conjunction with tryptic digests, HTA-Proteases offer increased proteome coverage, unique peptide targets, and brief one-step protocols amenable to automation, rapid turnaround, and high-throughput approaches.
Topics: Peptide Hydrolases; Trypsin; Proteome; Proteomics; Workflow; Peptides; Membrane Proteins
PubMed: 37634627
DOI: 10.1016/j.jprot.2023.104992 -
Foods (Basel, Switzerland) Mar 2023Papain, bromelain, and ficin are commonly used plant proteases used for meat tenderization. Other plant proteases explored for meat tenderization are actinidin,... (Review)
Review
Papain, bromelain, and ficin are commonly used plant proteases used for meat tenderization. Other plant proteases explored for meat tenderization are actinidin, zingibain, and cucumin. The application of plant crude extracts or powders containing higher levels of compounds exerting tenderizing effects is also gaining popularity due to lower cost, improved sensory attributes of meat, and the presence of bioactive compounds exerting additional benefits in addition to tenderization, such as antioxidants and antimicrobial effects. The uncontrolled plant protease action could cause excessive tenderization (mushy texture) and poor quality due to an indiscriminate breakdown of proteins. The higher cost of separation and the purification of enzymes, unstable structure, and poor stability of these enzymes due to autolysis are some major challenges faced by the food industry. The meat industry is targeting the recycling of enzymes and improving their stability and shelf-life by immobilization, encapsulation, protein engineering, medium engineering, and stabilization during tenderization. The present review critically analyzed recent trends and the prospects of the application of plant proteases in meat tenderization.
PubMed: 36981262
DOI: 10.3390/foods12061336 -
Current Issues in Molecular Biology 2019Oral streptococci depend on two-component signal transduction systems (TCSs), the most widespread regulatory systems in bacteria, to detect and respond to diverse... (Review)
Review
Oral streptococci depend on two-component signal transduction systems (TCSs), the most widespread regulatory systems in bacteria, to detect and respond to diverse stresses in oral environment. Among the larger panel of TCSs equipped by oral streptococci, ComDE TCS is thought to be one of the most classical TCSs. So far, it has been proved that ComDE TCS could play critical roles in environmental stress responses to acid, antibiotic, oxidative pressures and so on, and modulating multiple virulence traits like biofilm formation, bacteriocin production, competence, autolysis. Here, the well characterized and are chosen as the representative species to introduce the composition, signaling pathways and regulated phenotypes of ComDE TCS in oral streptococci. The potential ComDE TCS-targeted antimicrobial applications are also discussed at last.
Topics: Adaptation, Physiological; Bacterial Proteins; Biofilms; Dental Caries; Dietary Carbohydrates; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Histidine Kinase; Humans; Mouth; Oxidative Stress; Signal Transduction; Streptococcal Infections; Streptococcus mutans; Streptococcus pneumoniae; Virulence
PubMed: 31166173
DOI: 10.21775/cimb.032.201 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Mar 2019Recombinant bacterial vector vaccines have been widely used as carriers for the delivery of protective antigens and nucleic acid vaccines to prevent certain infectious... (Review)
Review
Recombinant bacterial vector vaccines have been widely used as carriers for the delivery of protective antigens and nucleic acid vaccines to prevent certain infectious diseases because of their ability to induce mucosal immunity, humoral immunity and cellular immunity. However, protective antigens and nucleic acids recombined into bacterial vector vaccines are difficult to be released into host cells because of the presence of bacterial cell wall. Vaccine strains that are residual in animals or livestock products may also cause environmental contamination and spread of the vaccine strains. The effective solution for these problems is to construct an auto-lysis system that can regulate the vaccine strains to grow normally in vitro while lysis in vivo. The lysis systems that have been applied in germs mainly include: the lysis system based on regulated delayed peptidoglycan synthesis, the lysis system based on the regulation of bacteriophage lysis protein and the lysis system based on the toxin-antitoxin system. In addition, a potential lysis system based on bacterial Type Ⅵ Secretion System (T6SS) is also expected to be a new method for the construction of auto-lysis strains. This review will focus on the regulatory mechanisms of these bacterial lysis systems.
Topics: Animals; Antigens, Bacterial; Bacterial Vaccines; Vaccines, Attenuated; Vaccines, DNA
PubMed: 30912346
DOI: 10.13345/j.cjb.180283 -
ELife Jan 2022The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and...
The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.
Topics: Bacterial Proteins; Cell Wall; Endopeptidases; Peptidoglycan; Periplasm; Vibrio cholerae
PubMed: 35073258
DOI: 10.7554/eLife.73178 -
Scientific Reports Mar 2017In contrast to many nanotoxicity studies where nanoparticles (NPs) are observed to be toxic or reduce viable cells in a population of bacteria, we observed that...
In contrast to many nanotoxicity studies where nanoparticles (NPs) are observed to be toxic or reduce viable cells in a population of bacteria, we observed that increasing concentration of TiO NPs increased the cell survival of Bacillus subtilis in autolysis-inducing buffer by 0.5 to 5 orders of magnitude over an 8 hour exposure. Molecular investigations revealed that TiO NPs prevent or delay cell autolysis, an important survival and growth-regulating process in bacterial populations. Overall, the results suggest two potential mechanisms for the disruption of autolysis by TiO NPs in a concentration dependent manner: (i) directly, through TiO NP deposition on the cell wall, delaying the collapse of the protonmotive-force and preventing the onset of autolysis; and (ii) indirectly, through adsorption of autolysins on TiO NP, limiting the activity of released autolysins and preventing further lytic activity. Enhanced darkfield microscopy coupled to hyperspectral analysis was used to map TiO deposition on B. subtilis cell walls and released enzymes, supporting both mechanisms of autolysis interference. The disruption of autolysis in B. subtilis cultures by TiO NPs suggests the mechanisms and kinetics of cell death may be influenced by nano-scale metal oxide materials, which are abundant in natural systems.
Topics: Adsorption; Bacillus subtilis; Bacteriolysis; Cell Wall; Colony Count, Microbial; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Metal Nanoparticles; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Proton-Motive Force; Titanium
PubMed: 28303908
DOI: 10.1038/srep44308 -
Scientific Reports Feb 2022Coprinus comatus, widely known as "Jituigu", is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of...
Coprinus comatus, widely known as "Jituigu", is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of the fruiting bodies after harvest. To gain insight into the molecular mechanism underlying this autolysis, we divided the growth of C. comatus fruiting bodies into four stages: infant stage (I), mature stage (M), discolored stage (D), and autolysis stage (A). We then subjected these stages to de novo transcriptomic analysis using high-throughput Illumina sequencing. A total of 12,946 unigenes were annotated and analyzed with the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). We analyzed the differentially expressed genes (DEGs) between stages I and M, M and D, and D and A. Because the changes from M to D are thought to be related to autolysis, we focused on the DEGs between these two stages. We found that the pathways related to metabolic activity began to vary in the transition from M to D, including pathways named as autophagy-yeast, peroxisome, and starch and sucrose metabolism. This study also speculates the possible process of the autolysis of Coprinus comatus. In addition, 20 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study, which is the first to describe the transcriptome of C. comatus, provides a foundation for future studies concerning the molecular basis of the autolysis of its fruiting bodies.
Topics: China; Coprinus; Food; Fruiting Bodies, Fungal; Gene Expression Profiling; Gene Ontology; Genes, Fungal; High-Throughput Nucleotide Sequencing; Metabolic Networks and Pathways; Real-Time Polymerase Chain Reaction; Transcriptome
PubMed: 35169137
DOI: 10.1038/s41598-022-06103-z -
Microorganisms May 2022Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of...
Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains- BL23 and GG-in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.
PubMed: 35744660
DOI: 10.3390/microorganisms10061142 -
Frontiers in Microbiology 2016Vancomycin has been used as the last resort in the clinical treatment of serious infections. Vancomycin-intermediate (VISA) was discovered almost two decades ago.... (Review)
Review
Vancomycin has been used as the last resort in the clinical treatment of serious infections. Vancomycin-intermediate (VISA) was discovered almost two decades ago. Aside from the vancomycin-intermediate phenotype, VISA strains from the clinic or laboratory exhibited common characteristics, such as thickened cell walls, reduced autolysis, and attenuated virulence. However, the genetic mechanisms responsible for the reduced vancomycin susceptibility in VISA are varied. The comparative genomics of vancomycin-susceptible (VSSA)/VISA pairs showed diverse genetic mutations in VISA; only a small number of these mutations have been experimentally verified. To connect the diversified genotypes and common phenotypes in VISA, we reviewed the genetic alterations in the relative determinants, including mutations in the , and genes. Especially, we analyzed the mechanism through which diverse mutations mediate vancomycin resistance. We propose a unified model that integrates diverse gene functions and complex biochemical processes in VISA upon the action of vancomycin.
PubMed: 27790199
DOI: 10.3389/fmicb.2016.01601 -
Current Opinion in Plant Biology Feb 2017Plant development requires specific cells to be eliminated in a predictable and genetically regulated manner referred to as programmed cell death (PCD). However, the... (Review)
Review
Plant development requires specific cells to be eliminated in a predictable and genetically regulated manner referred to as programmed cell death (PCD). However, the target cells do not merely die but they also undergo autolysis to degrade their cellular corpses. Recent progress in understanding developmental cell elimination suggests that distinct proteins execute PCD sensu stricto and autolysis. In addition, cell death alone and cell dismantlement can fulfill different functions. Hence, it appears biologically meaningful to distinguish between the modules of PCD and autolysis during plant development.
Topics: Apoptosis; Autophagy; Plant Development
PubMed: 27936412
DOI: 10.1016/j.pbi.2016.11.017