Specific Cleavage Research Articles

Mycobacterium smegmatis putative Holliday junction resolvases RuvC and RuvX play complementary roles in the processing of branched DNA structures

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the E. coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and double-stranded DNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities, and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and single-stranded DNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis.

Synthesis and Properties of RNA Modified with Thioamide Internucleoside Linkage.

Recent success of RNA therapeutics has reinvigorated interest in chemical modifications of RNA. As exemplified by the phosphorothioates, modifications of sugar-phosphate backbone have been remarkably impactful but relatively underexplored in therapeutic RNAs. The present study reports synthesis, thermal stability, and RNA interference activity of RNAs modified with thioamide linkages. Compared to the previously studied amide-modified RNA, thioamide linkages strongly destabilized a short self-complementary RNA model duplex. However, in short interfering RNAs amides and thioamides had a similar effect on duplex stability and target RNA cleavage activity and specificity. Hence, the thioamide may be added to the toolbox of chemical biologist as a useful backbone modification well tolerated by the RNA interference machinery.

Electrochemical detection of FTO with N3-kethoxal labeling and MazF cleavage.

N6-Methyladenosine (m6A) is a prevalent modification in eukaryotic mRNAs and is linked to various human cancers. The fat mass and obesity-associated protein (FTO), a key m6A demethylase, is crucial in m6A regulation, affecting many biological processes and diseases. Detecting FTO is vital for clinical and research applications. Our study leverages the specific cleavage properties of the MazF endoribonuclease to design an electrochemical method with signal amplification guided by streptavidin-horseradish peroxidase (SA-HRP), intended for FTO detection. Initially, the compound N3-kethoxal is employed for its reversible tagging ability, selectively attaching to guanine (G) bases. Subsequently, dibenzocyclooctyne polyethylene glycol biotin (DBCO-PEG4-Biotin), is introduced through a reaction with N3-kethoxal. HRP is then employed to catalyze the redox system to enhance the current response further. A promising linear correlation between the peak current and the FTO concentration was observed within the range of 7.90 × 10-8 to 3.50 × 10-7 M, with a detection limit of 5.80 × 10-8 M. Moreover, this method assessed the FTO inhibitor FB23's inhibitory effect, revealing a final IC50 value of 54.73 nM. This result aligns with the IC50 value of 60 nM obtained through alternative methods and is very close to the values reported in the literature. The study provides reference value for research into obesity, diabetes, cancer, and other FTO-related diseases, as well as for the screening of potential therapeutic drugs.

TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data.

State-of-the-art mass spectrometers combined with modern bioinformatics algorithms for peptide-to-spectrum matching (PSM) with robust statistical scoring allow for more variable features (i.e., post-translational modifications) being reliably identified from (tandem-) mass spectrometry data, often without the need for biochemical enrichment. Semi-specific proteome searches, that enforce a theoretical enzymatic digestion to solely the N- or C-terminal end, allow to identify of native protein termini or those arising from endogenous proteolytic activity (also referred to as "neo-N-termini" analysis or "N-terminomics"). Nevertheless, deriving biological meaning from these search outputs can be challenging in terms of data mining and analysis. Thus, we introduce TermineR, a data analysis approach for the (1) annotation of peptides according to their enzymatic cleavage specificity and known protein processing features, (2) differential abundance and enrichment analysis of N-terminal sequence patterns, and (3) visualization of neo-N-termini location. We illustrate the use of TermineR by applying it to tandem mass tag (TMT)-based proteomics data of a mouse model of polycystic kidney disease, and assess the semi-specific searches for biological interpretation of cleavage events and the variable contribution of proteolytic products to general protein abundance. The TermineR approach and example data are available as an R package at https://github.com/MiguelCos/TermineR.

A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage.

Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.

Architecture and activation mechanism of the bacterial PARIS defence system.

Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race1-5. While the mechanisms of many bacterial antiphage defence systems are known1, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.

Membrane Localization of RNase Y Is Important for Global Gene Expression in Bacillus subtilis.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

An ultrasensitive DNA-enhanced amplification method for detecting cfDNA drug-resistant mutations in non-small cell lung cancer with selective FEN-assisted degradation of dominant somatic fragments.

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5'-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing arapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors.

A Versatile β-Glycosidase from Petroclostridium xylanilyticum Prefers the Conversion of Ginsenoside Rb3 over Rb1, Rb2, and Rc to Rd by Its Specific Cleavage Activity toward 1,6-Glycosidic Linkages.

To convert ginsenosides Rb1, Rb2, Rb3, and Rc into Rd by a single enzyme, a putative β-glycosidase (Pxbgl) from the xylan-degrading bacterium Petroclostridium xylanilyticum was identified and used. The kcat/Km value of Pxbgl for Rb3 was 18.18 ± 0.07 mM-1/s, which was significantly higher than those of Pxbgl for other ginsenosides. Pxbgl converted almost all Rb3 to Rd with a productivity of 5884 μM/h, which was 346-fold higher than that of only β-xylosidase from Thermoascus aurantiacus. The productivity of Rd from the Panax ginseng root and Panax notoginseng leaf was 146 and 995 μM/h, respectively. Mutants N293 K and I447L from site-directed mutagenesis based on bioinformatics analysis showed an increase in specific activity of 29 and 7% toward Rb3, respectively. This is the first report of a β-glycosidase that can simultaneously remove four different glycosyls at the C-20 position of natural PPD-type ginsenosides and produce Rd as the sole product from P. notoginseng leaf extracts with the highest productivity.

CRISPR Based Gene Knock Out Study in Western Clawed Frog

The advancement of biotechnology has contributed a revolutionary change in field of gene editing for the medical research and industrial biotechnology. There are different types of technology developed in recent era but the contributions of CRISPR/Cas 9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) has the major role for the advancement of gene editing[1]. Anciently it was known as acquired immunity that protects the bacteria from plasmid infection. It switches the bacterial immune system to eliminate the odd genetic material[2]. The technology has enormous potential to edit the genomes very accurate and efficient manner. It also provides various applications in the field of medical research. There are several elements involved in the CRISPR process involved in the process of gene editing. The developments of the process increased its potential for various model organisms. The major two components of the process are generally the sgRNA(Synthetic Single guide RNA) and the Cas 9[3]. The sgRNA is associated with genome specific side targeting and the Cas 9 has the ability to form the side specific DNA cleavage. There are several advantages of this technology over the disease like cystic fibrosis, sickle cell anemia, hemophilia and single nucleotide based genetic disorders[4]

Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Modulation of diverse biological processes by CPSF, the master regulator of mRNA 3' ends.

The cleavage and polyadenylation specificity factor (CPSF) complex plays a central role in the formation of mRNA 3' ends, being responsible for the recognition of the poly(A) signal sequence, the endonucleolytic cleavage step, and recruitment of poly(A) polymerase. CPSF has been extensively studied for over three decades, and its functions and those of its individual subunits are becoming increasingly well-defined, with much current research focusing on the impact of these proteins on the normal functioning or disease/stress states of cells. In this review, we provide an overview of the general functions of CPSF and its subunits, followed by a discussion of how they exert their functions in a surprisingly diverse variety of biological processes and cellular conditions. These include transcription termination, small RNA processing, and R-loop prevention/resolution, as well as more generally cancer, differentiation/development, and infection/immunity.

A Developmental Mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage.

Alternative Cleavage and Polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3'UTRs from the same genetic locus, potentially impacting mRNA translation, localization and stability. Developmentally regulated APA can thus make major contributions to cell-type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, approximately 500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of Cleavage Factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knock down of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell-type-specific APA at selected genes.

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand readsand (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.

X-Ray powder diffraction - structure prediction of unknown stone using foolproof and GSAS - Rietveld Refinement

A slightly violetish blue, cone shaped, hexagonally soft sheen, a soft lustre on a surface, and semi/nearly transparent with reasonable weight of a natural stone has been analysed as a part of investigation for determining the components of the stone thereby leading to identify as to whether the stone is in a category of gemstone, radioactive elements, or otherwise. Philips, X-pert Pro, DY993 X-ray Powder Diffractometer has been employed for collecting the powder diffraction data in the Bragg-Brentano geometry using proportional counter in the 5° < 2θ < 85° range at room temperature 300 K. The data, thus, obtained from the XRPD has been used in FullProf and GSAS (General Structure Analysis Software) software with the Rietveld refinement technique for elucidating the possible molecular structure of the unknown stone. In addition to the XRPD technique, the physical and optical parameters such as specific gravity, hardness, luster, transparency, cleavage, streak and other associated minerals have also been measured. This study reveals that the stone is in a category of precious stone which is of mineral deposition Corundum, a blue sapphire. The structure has been determined from Rietveld analysis in space group R-3c, with a= 4.7655 (2) Å and c = 13.0132(3) Å.

A novel AIE-based mitochondria-targeting fluorescent probe for monitoring of the fluctuation of endogenous hypochlorous acid in ferroptosis models.

Ferroptosis is a way of cell death mainly due to the imbalance between the production and degradation of lipid reactive oxygen species, which is closely associated with various diseases. Endogenous hypochlorous acid (HOCl) mainly produced in mitochondria is regarded as an important signal molecule of ferroptosis. Therefore, monitoring the fluctuation of endogenous HOCl is beneficial to better understand and treat ferroptosis-related diseases. Inspired by the promising aggregation-induced emission (AIE) properties of tetraphenylethene (TPE), herein, we rationally constructed a novel AIE-based fluorescent probe, namely QTrPEP, for HOCl with nice mitochondria-targeting ability and high sensitivity and selectivity. Probe QTrPEP consisted of phenylborate ester and the AIE fluorophore of quinoline-conjugated triphenylethylene (QTrPE). HOCl can brighten the strong fluorescence through a specific HOCl-triggered cleavage of the phenylborate ester bond and release ofQTrPE, which has been demonstrated by MS, HPLC, and DLS experiments. In addition, combining QTrPE-doped test strips with a smartphone-based measurement demonstrated the excellent performance of the probe to sense HOCl. The obtained favorable optical properties and negligible cytotoxicity allowed the use of this probe for tracking of HOCl in three different cells. In particular, this work represents the first AIE-based mitochondria-targeting fluorescent probe for monitoring the fluctuation of HOCl in ferroptosis.

How D-amino acids embedded in the protein sequence modify its digestibility: Behaviour of digestive enzymes tested on a model peptide used as target

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH2 (belonging to β-lactoglobulin, position 48–60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved.

The nuclear localization signal of CPSF6 governs post-nuclear import steps of HIV-1 infection.

The early stages of HIV-1 infection include the trafficking of the viral core into the nucleus of infected cells. However, much remains to be understood about how HIV-1 accomplishes nuclear import and the consequences of the import pathways utilized on nuclear events. The host factor cleavage and polyadenylation specificity factor 6 (CPSF6) assists HIV-1 nuclear localization and post-entry integration targeting. Here, we used a CPSF6 truncation mutant lacking a functional nuclear localization signal (NLS), CPSF6-358, and appended heterologous NLSs to rescue nuclear localization. We show that some, but not all, NLSs drive CPSF6-358 into the nucleus. Interestingly, we found that some nuclear localized CPSF6-NLS chimeras supported inefficient HIV-1 infection. We found that HIV-1 still enters the nucleus in these cell lines but fails to traffic to speckle-associated domains (SPADs). Additionally, we show that HIV-1 fails to efficiently integrate in these cell lines. Collectively, our results demonstrate that the NLS of CPSF6 facilitates steps of HIV-1 infection subsequent to nuclear import and additionally identify the ability of canonical NLS sequences to influence cargo localization in the nucleus following nuclear import.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

Thermostable Bacterial Collagenolytic Proteases: A Review.

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the β-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.