SARS-CoV-2 3CL (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL.

SARS-CoV-2 3C-like protease (3CL or M) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL expression and viral replication. Unexpectedly, 3CL and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CL main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CL) is essential for protein excision from the viral polyprotein. 3CL inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CL from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS).

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL substrate degradome.

The main viral protease (3CL) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells.

Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis.

Background: Intestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear.

Methods: Chronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis.

Proteases and their inhibitors as prognostic factors for high-grade serous ovarian cancer.

Ovarian carcinoma is one of the most lethal malignancies, but only very few prognostic biomarkers are known. The degradome, comprising proteases, protease non-proteolytic homologues and inhibitors, have been involved in the prognosis of many cancer types, including ovarian carcinoma. The prognostic significance of the whole degradome family has not been specifically studied in high-grade serous ovarian cancer.

C-terminal truncation of IFN-γ inhibits proinflammatory macrophage responses and is deficient in autoimmune disease.

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages.

Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript.

Novel grooved substrata stimulate macrophage fusion, CCL2 and MMP-9 secretion.

Rough surface topographies on implants attract macrophages but the influence of topography on macrophage fusion to produce multinucleated giant cells (MGC) and foreign body giant cells (FBGC) is unclear. Two rough novel grooved substrata, G1 and G2, fabricated by anisotropic etching of Silicon <110> crystals without the use of photolithographic patterning, and a control smooth surface (Pol) were produced and replicated in epoxy. The surfaces were compared for their effects on RAW264.

Active site specificity profiling datasets of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9, 12, 13 and 14.

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis.

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries.

Macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis.

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time.

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries.

Background: Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.

Methodology/principal Finding: To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS).

Network analyses reveal pervasive functional regulation between proteases in the human protease web.

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized.

The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human.

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step.

Repression of Salmonella enterica phoP expression by small molecules from physiological bile.

Infection with Salmonella enterica serovar Typhi in humans causes the life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate.

Microarray and proteomic analysis of breast cancer cell and osteoblast co-cultures: role of osteoblast matrix metalloproteinase (MMP)-13 in bone metastasis.

Dynamic reciprocal interactions between a tumor and its microenvironment impact both the establishment and progression of metastases. These interactions are mediated, in part, through proteolytic sculpting of the microenvironment, particularly by the matrix metalloproteinases, with both tumors and stroma contributing to the proteolytic milieu. Because bone is one of the predominant sites of breast cancer metastases, we used a co-culture system in which a subpopulation of the highly invasive human breast cancer cell line MDA-MB-231, with increased propensity to metastasize to bone, was overlaid onto a monolayer of differentiated osteoblast MC3T3-E1 cells in a mineralized osteoid matrix.

Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates.

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development.

Matrix metalloproteinase 8 deficiency in mice exacerbates inflammatory arthritis through delayed neutrophil apoptosis and reduced caspase 11 expression.

Objective: Neutrophil accumulation is balanced by both cell infiltration and cell clearance, the controls of which are pivotal in the pathogenesis of rheumatoid arthritis (RA) and other chronic inflammatory diseases. Of the neutrophil-specific proteases, matrix metalloproteinase 8 (MMP-8; also known as neutrophil collagenase or collagenase 2) is traditionally viewed as being crucial for collagen degradation and hence cell migration and infiltration. This study was undertaken to examine the role of MMP-8 in a murine model of spontaneous RA.

Novel matrix metalloproteinase inhibitor [18F]marimastat-aryltrifluoroborate as a probe for in vivo positron emission tomography imaging in cancer.

Matrix metalloproteinases (MMP), strongly associated pathogenic markers of cancer, have undergone extensive drug development programs. Marimastat, a noncovalent MMP inhibitor, was conjugated with FITC to label cellular metalloproteinase cancer targets in MDA-MB-231 cells in vitro. Punctate localization of active transmembrane MMP14 was observed.

Proenzyme structure and activation of astacin metallopeptidase.

Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a "cysteine switch" mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion.

Analysis of the degradome with the CLIP-CHIP microarray.

The degradome microarray - CLIP-CHIP - is a dedicated and focused array that allows the analysis of all proteases, non-proteolytic homologs, and protease inhibitor gene transcripts in the human and murine genomes at the mRNA transcript level.Based on unique 70-mer oligonucleotides, designed to match parts of the sequence of known or predicted protease and inhibitor mRNAs in both species and printed on a glass-matrix surface, the CLIP-CHIP microarray can be used to analyze differentially expressed protease and inhibitor gene products and give expression profiles for any analyzed sample.

The CLIP-CHIP oligonucleotide microarray: dedicated array for analysis of all protease, nonproteolytic homolog, and inhibitor gene transcripts in human and mouse.

The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples.

The CLIP-CHIP oligonucleotide microarray: dedicated array for analysis of all protease, nonproteolytic homolog, and inhibitor gene transcripts in human and mouse.

The CLIP-CHIP oligonucleotide microarray allows the analysis of mRNA transcript levels in a tissue sample for all proteases, nonproteolytic homologs, and protease inhibitors of the human and mouse genome. In the protocol presented in this unit, total RNA is extracted from a tissue, and the resulting mRNA is reverse transcribed into cDNA and dsDNA and then amplified in an in vitro transcription reaction. The amplified antisense RNA is labeled with a fluorescent dye and hybridized to the CLIP-CHIP, which contains unique oligonucleotides that are specifically designed for the protease, nonproteolytic homologs, protease inhibitors, and control samples.

Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors.

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2.

Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing.

Astacin (EC 3.4.24.