Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer’s disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.
35Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is 36 prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the 37 coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host 38 specificity, but virus entry is a complex process requiring a concerted action of multiple cellular 39 elements. Here, we show that KLK13 is required for the infection of the human respiratory 40 epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. 41 We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving 42 that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that 43 protease distribution and specificity predetermines the tissue and cell specificity of the virus 44 and may also regulate interspecies transmission. It is also of importance that presented data may 45 be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand 46 the differences in their zoonotic potential. : bioRxiv preprint 48Coronaviruses are the largest group within the order Nidovirales. Mainly, they cause 49 respiratory and enteric diseases in humans and animals, but some can cause more serious 50 conditions such as hepatitis, peritonitis, or neurological disease. Seven coronaviruses infect 51 humans, four of which (human coronavirus [HCoV]-229E, HCoV-NL63, HCoV-OC43, and 52 HCoV-HKU1) cause relatively mild upper and lower respiratory tract disease and two (SARS-53 CoV and MERS-CoV) are associated with severe, life-threatening respiratory infections and 54 multiorgan failure (1-6). Furthermore, in December 2019 a novel coronavirus SARS-CoV-2 55 emerged in Hubei province, China, causing pneumonia. To date, almost 90,000 cases were 56 identified and 3,000 patients died worldwide. 57 Coronaviral infection is initiated by interaction between the trimeric spike (S) protein 58 and its receptor, which is expressed on the surface of the susceptible cell. A number of adhesion 59 and entry receptors have been described for coronaviruses. For example, HCoV-229E (similar 60 to many other alphacoronaviruses) utilizes aminopeptidase N (APN) as the primary entry port 61 (7). Surprisingly, its cousin HCoV-NL63 shares receptor specificity with the evolutionarily 62 distant SARS-CoV and SARS-CoV-2: all hijack angiotensin-converting enzyme 2 (ACE2) (8-63 11). HCoV-NL63 was also shown to use heparan sulfate as a primary attachment site (12-14). 64 A very different receptor is recognized by MERS-CoV, which binds to dipeptidyl-peptidase 4 65 (DPP4) (9, 15, 16). Another betacoronavirus, HCoV-OC43, binds to N-acetyl-9-O-66 acetylneuraminic acid (17, 18). HCoV-HKU1 remains the great unknown because its cellular 67 receptor has not been identified and all efforts to culture the virus in vitro have failed.68 HCoV-HKU1 was identified in Hong Kong in 2004. The virus was present in a sample 69 obtained from an elderly patient with severe pneumonia (...
Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration-and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.
Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.
Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.
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