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Fibrosis is a common manifestation of most progressive and degenerative diseases, with myofibroblast activation and matrix accumulation playing a key role. In this issue of the JCI, Hoeft et al. identify the important role of ADAMTS12 in fibroblast activation. ADAMTS12, a secreted protein, is involved in extracellular matrix (ECM) remodeling, cell signaling, and inflammation. ADAMTS12 facilitates proteolysis by cleaving various substrates such as ECM components, which are vital for cellular signaling and remodeling. Additionally, it modulates cell-matrix interactions, influencing cell adhesion and migration, and plays an important role in the inflammatory processes. Understanding the role of ADAMTS12 offers potential therapeutic insights for targeting fibrosis in progressive diseases., Fibrosis in heart and kidney disease Fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix (ECM) components, particularly collagen, in organs, including the heart and kidneys, often [...]

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury- responsive fibroblast subset defined by aberrant high JAK/STAT signaling., Introduction Fibrosis represents the uncontrolled replacement of parenchymal tissue by extracellular matrix (ECM) (1). Whereas deposition of ECM sustains tissue integrity immediately after injury, continued deposition of ECM by myofibroblasts [...]

Secretory leukocyte protease inhibitor (SLPI) is a small but powerful member of the serine protease inhibitor family, which includes proteins such as elafin and [alpha]1-antitrypsin. These proteins all have similar structures and antiprotease abilities, but SLPI has been found to have an additional role as an anti-inflammatory factor. It can inhibit the production of pro-inflammatory cytokines in cells stimulated with lipopolysaccharide, prevent neutrophil infiltration in murine models of lung and liver injury, and regulate the activity of the transcription factor NF-[KAPPA]B. In this review, we will revisit SLPI's unique biochemistry, and then explore how its anti-inflammatory functions can be linked to more recent findings showing that SLPI can localize to the nuclei of cells, bind DNA, and act as a regulator of gene expression. Key words: SLPI, inflammation, nucleus, promoter, transcription, NF-[KAPPA]B, cytokine, TNF[alpha]. La proteine SLPI (Secretory leukocyte protease inhibitor) est un membre de petite taille, mais puissant de la famille des inhibiteurs de protease a serine, qui comprend des proteines telles que l'elafine et l'[alpha]1-antitrypsine. Ces proteines ont toutes une structure et des capacites antiproteases similaires, mais on a decouvert que la SLPI exercait un role supplementaire en tant que facteur anti-inflammatoire. Elle peut inhiber la production de cytokines pro-inflammatoires dans des cellules stimulees par le lipopolysaccharide, prevenir l'infiltration de neutrophiles dans des modeles murins de lesions pulmonaires et hepatiques, et reguler l'activite du facteur de transcription NF-[KAPPA]B. Dans cette synthese, les auteurs reviendront sur la biochimieuniquedela SLPI, puis exploreront comment ses fonctions anti-inflammatoires peuvent etre liees a des decouvertes plus recentes montrant que la SLPI peut se localiser dans les noyaux des cellules, se lier a l'ADN et agir comme un regulateur de l'expression genique. [Traduit par la Redaction] Mots-cles: SLPI, inflammation, noyau, promoteur, transcription, NF-[kappa]B, cytokine, TNF[alpha]., Introduction SLPI protein structure and distribution The SLPI protein has many distinctive characteristics, the first being its extremely small size. SLPI is synthesized as a polypeptide chain comprising 132 amino [...]

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico, and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48 h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression. Key words: COVID-19, in silico, in vitro, pathway analysis, drug. La ribavirine est un analogue de la guanosine avec une activite antivirale de large spectre contre les virus a ARN. Sur cette base, nous avons tente de montrer l'activite anti-SARS-CoV-2 de cette molecule pharmacologique a l'aide de techniques in vitro, in silico et moleculaires. La ribavirine a presente une activite antivirale contre les cellules Vero E6 a la suite d'une infection par le SARS-CoV-2 tandis que le medicament lui-meme n'avait aucun effet toxique dans la plage de concentrations evaluee. L'etude in silico a laisse entrevoir que la ribavirine a un effet a large spectre sur le SARS-CoV- 2, avec une action sur differentes proteines virales. A l'aide des techniques moleculaires detaillees, nous avons montre que la ribavirine entraine une diminution de l'expression de TMPRSS2 en taux d'ARNm comme de proteines 48 h apres son administration. Nous avons observe que l'effet suppresseur de la ribavirine sur l'expression d'ACE2 en proteines etait dependant du type de cellules. Enfin, les epreuves sur l'activite proteolytique ont montre que la ribavirine presentait aussi un effet inhibiteur sur l'enzyme TMPRSS2. De ces resultats, nous avons tire l'hypothese selon laquelle la ribavirine pourrait permettre d'inhiber l'expression de TMPRSS2 par l'intermediaire d'une modulation de la formation de structures inhibitrices G-quadruplex au niveau du promoteur de TMPRSS2. En conclusion, la ribavirine est un medicament antiviral eventuel pour le traitement contre le SARS-CoV-2, et elle interfere avec l'expression de TMPRSS2 et d'ACE2. Mots-cles: COVID-19, in silico, in vitro, etude de voie de signalisation, medicament., Introduction The coronavirus 2019 (COVID-19) pandemic represents the strongest global public health threat since the influenza outbreak in 1918 and has rapidly resulted in significant health and economic burdens worldwide [...]

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy.sup.1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes.sup.5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy.sup.7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors. Fertilized Arabidopsis egg cells secrete endopeptidases into the extracellular space that cleave the pollen tube attractor LURE1, preventing polytubey., Author(s): Xiaobo Yu [sup.1] , Xuecheng Zhang [sup.1] , Peng Zhao [sup.1] , Xiongbo Peng [sup.1] , Hong Chen [sup.1] , Andrea Bleckmann [sup.2] , Anastasiia Bazhenova [sup.2] , Ce [...]

Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly.sup.1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions.sup.3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting-feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes. mTORC1 stabilizes HMG-CoA reductase, a rate-limiting enzyme in the cholesterol biosynthesis pathway, via the deubiquitylase USP20 in response to feeding., Author(s): Xiao-Yi Lu [sup.1] , Xiong-Jie Shi [sup.1] , Ao Hu [sup.1] , Ju-Qiong Wang [sup.1] , Yi Ding [sup.1] , Wei Jiang [sup.1] , Ming Sun [sup.1] , Xiaolu [...]

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread.sup.1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses.sup.3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-[kappa]B pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity. Biochemical, structural and functional studies on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease PLpro reveal that it regulates host antiviral responses by preferentially cleaving the ubiquitin-like interferon-stimulated gene 15 protein (ISG15) and identify this protease as a potential therapeutic target for coronavirus disease 2019 (COVID-19)., Author(s): Donghyuk Shin [sup.1] [sup.2] [sup.3] , Rukmini Mukherjee [sup.1] [sup.2] , Diana Grewe [sup.2] , Denisa Bojkova [sup.4] , Kheewoong Baek [sup.5] , Anshu Bhattacharya [sup.1] [sup.2] , Laura [...]

During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8-KO pups (Casp-[8.sup.ECKO]) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-[8.sup.ECKO] pups. Taking these data together, we show that Casp-8 acts in a cell death-independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP., Introduction Functional blood vessels are of vital importance. Impaired vessel formation contributes to many pathological situations, including ischemic and inflammatory disorders. Ischemic retinopathies are the main causes of severe visual [...]

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at [IQAD.sup.198][down arrow]S located at the intersubunit linker. Previously, weidentified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters ([k.sub.cat]/[K.sub.M]) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation. Key words: apoptosis, caspases, PEST motif, protease activation mechanism. Resume:Lepolypeptide zymogene procaspase-7 est forme d'un court pro-domaine, d'une grande sous-unite (p20)etd'une petite sous-unite (p10) liees par un connecteur inter-domaine. La caspase-7 est activee par les caspases initiatrices-8 et -9 ou par autocatalyse a la suite du clivage specifique a la position [IQAD.sup.198][down arrow]S localisee dans le connecteur inter-domaine. Precedemment, les auteurs avaient montre que des regions PEST faites des acides amines Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), et Gln (Q) sont des residus d'acides amines conserves flanquant les sites de clivage a l'interieur du pro-domaine et du connecteur inter-domaine chez tous les membres de la famille des caspases. Ils ont teste ici l'impact d'une substitution par une alanine d'acides amines des regions PEST sur l'auto-activation proteolytique de la procaspase-7 directement chez Escherichia coli. Le clivage des sous-unites p20 et p10 etait significativement retarde significativement chez les doubles mutants du pro-domaine (N18A/P26A) et du connecteur inter-domaine (S199A/P201A) de la caspase-7 comparativement a la caspase-7 sauvage. Les mutations S199A/P201A inhibaient effectivement le clivage de la petite sous-unite p10. Cependant, les mutations n'affectaient pas les parametres cinetiques ([k.sub.cat]/[K.sub.M]) et la specificite optimale pour le tetra-peptide (DEVD) de l'enzyme mutante purifiee. Les resultats suggerent que les acides amines PEST jouent un role dans le mecanisme moleculaire du clivage du pro-domaine et entre les sous-unites, et dans l'auto-activation de la caspase-7. [Traduit par la Redaction] Mots-cles : apoptose, caspases, motif PEST, mecanisme d'activation., Introduction Caspases belong to an evolutionarily conserved family of 14 cysteine aspartyl-specific proteases that initiate and induce signaling pathways for apoptotic cell death, inflammation, and cell differentiation (Earnshaw et al. [...]

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpCClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpCClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpCClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitinproteasome system., Author(s): Dbora Broch Trentini [1]; Marcin Jzef Suskiewicz [1]; Alexander Heuck [1]; Robert Kurzbauer [1]; Luiza Deszcz [1]; Karl Mechtler [1, 2]; Tim Clausen (corresponding author) [1] Proteins destined for [...]