Capturing a crucial 'disorder-to-order transition' at the heart of the coronavirus molecular pathology – triggered by highly persistent, interchangeable salt-bridges

The COVID-19 origin debate has greatly been influenced by Genome comparison studies of late, revealing the seemingly sudden emergence of the Furin-Like Cleavage Site at the S1/S2 junction of the SARS-CoV-2 Spike (FLCS_Spike) containing its 681_PRRAR_685 motif, absent in other related respiratory viruses. Being the rate-limiting (i.e., the slowest) step, the host Furin cleavage is instrumental in the abrupt increase in transmissibility in COVID-19, compared to earlier onsets of respiratory viral diseases. In such a context, the current paper entraps a disorder-to-order transition of the FLCS_Spike (concomitant to an entropy arrest) upon binding to Furin. The interaction clearly seems to be optimized for a more efficient proteolytic cleavage in SARS-CoV-2. The study further shows the formation of dynamically interchangeable and persistent networks of salt-bridges at the Spike-Furin interface in SARS-CoV-2 involving the three arginines (R682, R683, R685) of the FLCS_Spike with several anionic residues (E230, E236, D259, D264, D306) coming from Furin, strategically distributed around its catalytic triad. Multiplicity and structural degeneracy of plausible salt-bridge network archetypes seems the other key characteristic features of the Spike-Furin binding in SARS-CoV-2 allowing the system to breathe - a trademark of protein disorder transitions. Interestingly, with respect to the homologous interaction in SARS-CoV (2002/2003) taken as a baseline, the Spike-Furin binding events generally in the coronavirus lineage seems to have a preference for ionic bond formation, even with lesser number of cationic residues at their potentially polybasic FLCS_Spike patches. The interaction energies are suggestive of a characteristic metastabilities attributed to Spike-Furin interactions generally to the coronavirus lineage - which appears to be favorable for proteolytic cleavages targeted at flexible protein loops. The current findings not only offer novel mechanistic insights into the coronavirus molecular pathology and evolution but also add substantially to the existing theories of proteolytic cleavages.

Synergistic inhibition of two host factors that facilitate entry of Severe Acute Respiratory Syndrome Coronavirus 2

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ~5-10 fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ~5 fold or more decrease of half maximal effective concentrations (EC50 values) suggests a plausible treatment strategy based on the combined use of these inhibitors.

Stress-induced activation of receptor signaling by protease-mediated cleavage

Plants encode a large number of proteases in activating intracellular signaling through proteolytic cleavages of various protein substrates. One type of the substrates is proligands, including peptide hormones, which are perceived by cell surface-resident receptors. The peptide hormones are usually first synthesized as propeptides, and then cleaved by specific proteases for activation. Accumulating evidence indicates that the protease-mediated cleavage of proligands can be triggered by environmental stresses and subsequently activates plant stress signaling. In this perspective, we highlight several recent publications and provide an update about stress-induced cleavage of propeptides and receptor-associated components by proteases in the activation of cell surface-resident receptor signaling in plants. We also discuss some questions and future challenges in the research of protease functions in plant stress response.

Proteolytic Cleavages in the VEGF Family: Generating Diversity among Angiogenic VEGFs, Essential for the Activation of Lymphangiogenic VEGFs

Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph­angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the “mature” growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.

Proteolytic Regulation of the Lectin-Like Oxidized Lipoprotein Receptor LOX-1

The lectin-like oxidized-LDL (oxLDL) receptor LOX-1, which is broadly expressed in vascular cells, represents a key mediator of endothelial activation and dysfunction in atherosclerotic plaque development. Being a member of the C-type lectin receptor family, LOX-1 can bind different ligands, with oxLDL being the best characterized. LOX-1 mediates oxLDL uptake into vascular cells and by this means can promote foam cell formation. In addition, LOX-1 triggers multiple signaling pathways, which ultimately induce a pro-atherogenic and pro-fibrotic transcriptional program. However, the molecular mechanisms underlying this signal transduction remain incompletely understood. In this regard, proteolysis has recently emerged as a regulatory mechanism of LOX-1 function. Different proteolytic cleavages within the LOX-1 protein can initiate its turnover and control the cellular levels of this receptor. Thereby, cleavage products with individual biological functions and/or medical significance are produced. Ectodomain shedding leads to the release of a soluble form of the receptor (sLOX1) which has been suggested to have diagnostic potential as a biomarker. Removal of the ectodomain leaves behind a membrane-bound N-terminal fragment (NTF), which despite being devoid of the ligand-binding domain is actively involved in signal transduction. Degradation of this LOX-1 NTF, which represents an athero-protective mechanism, critically depends on the aspartyl intramembrane proteases Signal peptide peptidase-like 2a and b (SPPL2a/b). Here, we present an overview of the biology of LOX-1 focusing on how proteolytic cleavages directly modulate the function of this receptor and, what kind of pathophysiological implications this has in cardiovascular disease.

The Proteolytic Activation of Vascular Endothelial Growth Factors

Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph­angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. The activating cleavage of VEGF-C is mediated by at least five different proteases: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the "mature" growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by different proteases. During embryonic development, ADAMTS3 activates VEGF-C. In contrast, thrombin and plasmin likely activate VEGF-C/-D during tissue injury-induced lymphangiogenesis, and PSA and cathepsin D perhaps during tumor-associated pathological lymphangio­genesis. Additionally, cathepsin D from saliva might activate latent VEGF-C/-D upon wound licking, thereby accelerating healing. Similar to tyrosine kinase receptors and VEGFs themselves, these activating proteases could be targeted to modulate angiogenesis and lymphangiogenesis in relevant diseases.

Mouse models of human proprotein convertase insufficiency

The kexin-like proprotein convertases perform the initial proteolytic cleavages that ultimately generate a variety of different mature peptide and proteins, ranging from brain neuropeptides, to endocrine peptide hormones, to structural proteins, among others. In this review, we present a general introduction to proprotein convertase structure and biochemistry, followed by a comprehensive discussion of each member of the kexin-like subfamily of proprotein convertases. We summarize current knowledge of human proprotein convertase insufficiency syndromes, including genome-wide analyses of convertase polymorphisms, and compare these to convertase null and mutant mouse models. These mouse models have illuminated our understanding of the roles specific convertases play in human disease and have led to the identification of convertase-specific substrates; for example, the identification of pro-corin as a specific PACE4 substrate in the heart. We also discuss the limitations of mouse null models in interpreting human disease, such as differential precursor cleavage due to species-specific sequence differences, and the challenges presented by functional redundancy among convertases in attempting to assign specific cleavages and/or physiological roles. However, in most cases, knockout mouse models have added substantively both to our knowledge of diseases caused by human proprotein convertase insufficiency and to our appreciation of their normal physiological roles, as clearly seen in the case of the furin, PC1/3, and PC5/6 mouse models. The creation of more sophisticated mouse models with tissue- or temporally-restricted expression of specific convertases will improve our understanding of human proprotein convertase insufficiency and potentially provide support for the emerging concept of therapeutic inhibition of convertases.

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

Processing and Maturation of Cathepsin C Zymogen: A Biochemical and Molecular Modeling Analysis

Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon–Lefèvre and Haim–Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.

Dronc-independent basal executioner caspase activity sustains Drosophila imaginal tissue growth

Caspase is best known as an enzyme involved in programmed cell death, which is conserved among multicellular organisms. In addition to its role in cell death, caspase is emerging as an indispensable enzyme in a wide range of cellular functions, which have recently been termed caspase-dependent nonlethal cellular processes (CDPs). In this study, we examined the involvement of cell death signaling in tissue-size determination using Drosophila wing as a model. We found that the Drosophila executioner caspases Dcp-1 and Decay, but not Drice, promoted wing growth independently of apoptosis. Most of the reports on CDPs argue the importance of the spatiotemporal regulation of the initiator caspase, Dronc; however, this sublethal caspase function was independent of Dronc, suggesting a more diverse array of CDP regulatory mechanisms. Tagging of TurboID, an improved promiscuous biotin ligase that biotinylates neighboring proteins, to the C terminus of caspases revealed the differences among the neighbors of executioner caspases. Furthermore, we found that the cleavage of Acinus, a substrate of the executioner caspase, was important in promoting wing growth. These results demonstrate the importance of executioner caspase-mediated basal proteolytic cleavage of substrates in sustaining tissue growth. Given the existence of caspase-like DEVDase activity in a unicellular alga, our results likely highlight the original function of caspase—not cell death, but basal proteolytic cleavages for cell vigor.