Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

Inhibition of mannan-binding lectin associated serine protease (MASP)-2 reduces the cognitive deficits in a mouse model of severe traumatic brain injury.

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI.

The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types.

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.

Serine protease inhibitor, SerpinA3n, regulates cardiac remodelling after myocardial infarction.

AIMS: Following myocardial infarction (MI), the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix (ECM) by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure, and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors, and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human MI as well as non-ischaemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodelling is poorly understood. The aim of this study was to understand the role of Serpins in regulating scar formation after MI. METHODS AND RESULTS: Using a SerpinA3n conditional knockout mice model, we observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homologue of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix, and significantly worse post-infarct cardiac function. Single-cell transcriptomics complemented with histology in SerpinA3n-deficient animals demonstrated increased inflammation, adverse myocyte hypertrophy, and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross-linking of ECM peptides consistent with increased proteolysis in SerpinA3n-deficient animals. CONCLUSION: Our study demonstrates a hitherto unappreciated causal role of Serpins in regulating matrix function and post-infarct cardiac remodelling.

Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Kallikrein-related peptidase's significance in Alzheimer's disease pathogenesis: A comprehensive survey.

Alzheimer's disease (AD) and related dementias constitute an important global health challenge. Detailed understanding of the multiple molecular mechanisms underlying their pathogenesis constitutes a clue for the management of the disease. Kallikrein-related peptidases (KLKs), a lead family of serine proteases, have emerged as potential biomarkers and therapeutic targets in the context of AD and associated cognitive decline. Hence, KLKs were proposed to display multifaceted impacts influencing various aspects of neurodegeneration, including amyloid-beta aggregation, tau pathology, neuroinflammation, and synaptic dysfunction. We propose here a comprehensive survey to summarize recent findings, providing an overview of the main kallikreins implicated in AD pathophysiology namely KLK8, KLK6 and KLK7. We explore the interplay between KLKs and key AD molecular pathways, shedding light on their significance as potential biomarkers for early disease detection. We also discuss their pertinence as therapeutic targets for disease-modifying interventions to develop innovative therapeutic strategies aimed at halting or ameliorating the progression of AD and associated dementias.

Covalent activity-based probes for imaging of serine proteases.

Serine proteases are one of the largest mechanistic classes of proteases. They regulate a plethora of biochemical pathways inside and outside the cell. Aberrant serine protease activity leads to a wide variety of human diseases. Reagents to visualize these activities can be used to gain insight into the biological roles of serine proteases. Moreover, they may find future use for the detection of serine proteases as biomarkers. In this review, we discuss small molecule tools to image serine protease activity. Specifically, we outline different covalent activity-based probes and their selectivity against various serine protease targets. We also describe their application in several imaging methods.

The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 in vivo.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells.

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl- current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Toxicological analyses of the venoms of Nigerian vipers Echis ocellatus and Bitis arietans.

BACKGROUND: Among the medically important snakes in Nigeria, Echis ocellatus and Bitis arietans have the most lethal venom. These venoms were classified according to the presence of snake venom metalloproteinases (SVMPs), snake venom phospholipase A2 (PLA2s), and snake venom serine proteases (SVSPs). Toxicological analyzes were performed to understand the significance of different protein families in venoms. METHODS: Proteins were separated from venom using column chromatography. The skin and footpad of mice were used to determine hemorrhagic and edematogenic activities. Caprine blood plasma was used to test fibrinolytic activity in vitro. RESULTS: The results showed that, compared to the crude venom, the SVMP fraction induced hemorrhagic effects with a diameter of 26.00 ± 1.00 mm in E. ocellatus and 21.33 ± 1.52 mm in B. arietans. Both SVSP and SVMP had anticoagulant effects; however, the SVSP fraction had a stronger effect, with a longer anticoagulation time of 30.00 ± 3.00 min in E. ocellatus and 26.00 ± 2.00 min in B. arietans. These main venom toxins, SVMPs, SVSPs, and PLA2, were found to have edema-forming effects that were optimal at 2 h after envenomation. PLA2s had the highest edema-inducing activity, with onset 30 min after envenomation. CONCLUSIONS: Given the importance of SVMPs in altering the integrity of the membrane structure and impairing the blood coagulation system, an antivenom that can specifically neutralize its activity could inhibit the hemorrhage effects of the venoms.

Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection.

Streptococcus suis (S. suis), a significant zoonotic bacterial pathogen impacting swine and human, is associated with severe systemic diseases such as streptococcal toxic shock-like syndrome, meningitis, septicaemia, and abrupt fatality. The multifaceted roles of complement components C5a and C3a extend to orchestrating inflammatory cells recruitment, oxidative burst induction, and cytokines release. Despite the pivotal role of subtilisin-like serine proteases in S. suis pathogenicity, their involvement in immune evasion remains underexplored. In the present study, we identify two cell wall-anchored subtilisin-like serine proteases in S. suis, SspA-1 and SspA-2, as binding partners for C3a and C5a. Through Co-Immunoprecipitation, Enzyme-Linked Immunosorbent and Far-Western Blotting Assays, we validate their interactions with the aforementioned components. However, SspA-1 and SspA-2 have no cleavage activity against complement C3a and C5a performed by Cleavage assay. Chemotaxis assays reveal that recombinant SspA-1 and SspA-2 effectively attenuate monocyte chemotaxis towards C3a and C5a. Notably, the ΔsspA-1, ΔsspA-1, and ΔsspA-1/2 mutant strains exhibit compromised survival in blood, and resistance of opsonophagocytosis, alongside impaired survival in blood and in vivo colonization compared to the parental strain SC-19. Critical insights from the murine and Galleria mellonella larva infection models further underscore the significance of sspA-1 in altering mortality rates. Collectively, our findings indicate that SspA-1 and SspA-2 are novel binding proteins for C3a and C5a, thereby shedding light on their pivotal roles in S. suis immune evasion and the pathogenesis.

Antiplatelet mechanism of a subtilisin-like serine protease from Solanum tuberosum (StSBTc-3).

The aims of this study are to characterize the antiplatelet activity of StSBTc-3, a potato serine protease with fibrino (geno) lytic activity, and to provide information on its mechanism of action. The results obtained show that StSBTc-3 inhibits clot retraction and prevents platelet aggregation induced by thrombin, convulxin, and A23187. Platelet aggregation inhibition occurs in a dose-dependent manner and is not affected by inactivation of StSBTc-3 with the inhibitor of serine proteases phenylmethylsulfonyl fluoride (PMSF). In addition, StSBTc-3 reduces fibrinogen binding onto platelets. In-silico calculations show a high binding affinity between StSBTc-3 and human α2bß3 integrin suggesting that the antiplatelet activity of StSBTc-3 could be associated with the fibronectin type III domain present in its amino acid sequence. Binding experiments show that StSBTc-3 binds to α2bß3 preventing the interaction between α2bß3 and fibrinogen and, consequently, inhibiting platelet aggregation. StSBTc-3 represents a promising compound to be considered as an alternative to commercially available drugs used in cardiovascular therapies.

In silico and in vitro elastase inhibition assessment assays of rosmarinic acid natural product from Rosmarinus officinalis Linn.

The use of various herbs and their compounds has been a strategy widely used in the fight against various human diseases. For example, rosmarinic acid, a bioactive phenolic compound commonly found in Rosemary plants (Rosmarinus officinalis Labiatae), has multiple therapeutic benefits in different diseases, such as cancer. Therefore, the study aimed to evaluate in silico and in vitro the inhibition potential of the enzyme Elastase from the porcine pancreas by rosmarinic acid isolated from the plant species R. officinalis Linn. Through Molecular Docking, the mechanism of action was investigated. In addition, rosmarinic acid presented a range of 5-60 µg/mL and significantly inhibited Elastase. At 60 µg/mL, there was an inhibition of 55% on the enzymatic activity. The results demonstrate the inhibition of Elastase by rosmarinic acid, which can lead to the development of new enzyme inhibitors that can be an inspiration for developing various drugs, including anticancer drugs.

Plasma levels of mannan-binding lectin-associated serine proteases are increased in type 1 diabetes patients with insulin resistance.

Activation of the lectin pathway of the complement system, as demonstrated by elevated levels of mannan-binding lectin proteins (MBL), contributes to vascular pathology in type 1 diabetes (T1D). Vascular complications are greatest in T1D individuals with concomitant insulin resistance (IR), however, whether IR amplifies activiation of the lectin pathway in T1D is unknown. We pooled pretreatment data from two RCTs and performed a cross-sectional analysis on 46 T1D individuals. We employed estimated glucose disposal rate (eGDR), a validated IR surrogate with cut-points of: <5.1, 5.1-8.7, and > 8.7 mg/kg/min to determine IR status, with lower eGDR values conferring higher degrees of IR. Plasma levels of MBL-associated proteases (MASP-1, MASP-2, and MASP-3) and their regulatory protein MAp44 were compared among eGDR classifications. In a subset of 14 individuals, we assessed change in MASPs and MAp44 following improvement in IR. We found that MASP-1, MASP-2, MASP-3, and MAp44 levels increased in a stepwise fashion across eGDR thresholds with elevated MASPs and MAp44 levels conferring greater degrees of IR. In a subset of 14 patients, improvement in IR was associated with significant reductions in MASPs, but not MAp44, levels. In conclusion, IR in T1D amplifies levels of MASP-1/2/3 and their regulator MAp44, and improvement of IR normalizes MASP-1/2/3 levels. Given that elevated levels of these proteins contribute to vascular pathology, amplification of the lectin pathway of the complement system may offer mechanistic insight into the relationship between IR and vascular complications in T1D.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.