Endogenous Protease Inhibitors Research Articles

Associations between Serum Kallistatin Levels and Markers of Glucose Homeostasis, Inflammation, and Lipoprotein Metabolism in Patients with Type 2 Diabetes and Nondiabetic Obesity.

Kallistatin is an endogenous serine proteinase inhibitor with various functions, including antioxidative, anti-inflammatory, and anti-atherosclerotic properties. To date, associations between kallistatin and lipoprotein subfractions are poorly investigated. In this study, we enrolled 62 obese patients with type 2 diabetes (T2D), 106 nondiabetic obese (NDO) subjects matched in gender, age, and body mass index, as well as 49 gender- and age-matched healthy, normal-weight controls. Serum kallistatin levels were measured with ELISA, and lipoprotein subfractions were analyzed using Lipoprint® (Quantimetrix Corp., Redondo Beach, CA, USA) gel electrophoresis. Kallistatin concentrations were significantly higher in T2D patients compared to NDO and control groups. We found significant positive correlations between very-low-density lipoprotein (VLDL), small high-density lipoprotein (HDL) subfractions, glucose, hemoglobin A1c (HbA1c), betatrophin, and kallistatin, while negative correlations were detected between mean low-density lipoprotein (LDL) size, large and intermediate HDL subfractions, and kallistatin in the whole study population. The best predictor of kallistatin was HbA1c in T2D patients, high-sensitivity C-reactive protein (hsCRP) and betatrophin in NDO patients, and hsCRP in controls. Our results indicate that kallistatin expression might be induced by persistent hyperglycemia in T2D, while in nondiabetic subjects, its production might be associated with systemic inflammation. The correlation of kallistatin with lipid subfractions may suggest its putative role in atherogenesis.

Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors.

Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Restriction of Viral Glycoprotein Maturation by Cellular Protease Inhibitors.

The human genome is estimated to encode more than 500 proteases performing a wide range of important physiological functions. They digest proteins in our food, determine the activity of hormones, induce cell death and regulate blood clotting, for example. During viral infection, however, some proteases can switch sides and activate viral glycoproteins, allowing the entry of virions into new target cells and the spread of infection. To reduce unwanted effects, multiple protease inhibitors regulate the proteolytic processing of self and non-self proteins. This review summarizes our current knowledge of endogenous protease inhibitors, which are known to limit viral replication by interfering with the proteolytic activation of viral glycoproteins. We describe the underlying molecular mechanisms and highlight the diverse strategies by which protease inhibitors reduce virion infectivity. We also provide examples of how viruses evade the restriction imposed by protease inhibitors. Finally, we briefly outline how cellular protease inhibitors can be modified and exploited for therapeutic purposes. In summary, this review aims to summarize our current understanding of cellular protease inhibitors as components of our immune response to a variety of viral pathogens.

Therapeutic potential of elafin in airway inflammatory disease

Elafin, which is derived from trappin-2 or pre-elafin by proteolysis, is an endogenous serine protease inhibitor with a low molecular weight. Its inhibitory activity is dependent on anchoring to the extracellular matrix by forming covalent bonds with its distinctive N-terminal domain via tissue transglutaminases. In addition to inhibiting proteases, it also exhibits anti-inflammatory, antibiotic, antifungal, antiviral and immunomodulatory functions. Elafin plays an important role in inflammatory disease and is a promising candidate for the anti-inflammatory treatment of respiratory diseases. This review will discuss the therapeutic potential of elafin in airway inflammation and provide evidence and suggestions for the future treatment of airway inflammatory diseases. In addition, the therapeutic potential of elafin in lung cancer is also discussed.

Eosinophilic Chronic Rhinosinusitis and Pathogenic Role of Protease.

Chronic rhinosinusitis (CRS) is an inflammation of the nasal and paranasal sinus mucosa, and eosinophilic CRS (eCRS) is a subtype characterized by significant eosinophil infiltration and immune response by T-helper-2 cells. The pathogenesis of eCRS is heterogeneous and involves various environmental and host factors. Proteases from external sources, such as mites, fungi, and bacteria, have been implicated in inducing type 2 inflammatory reactions. The balance between these proteases and endogenous protease inhibitors (EPIs) is considered important, and their imbalance can potentially lead to type 2 inflammatory reactions, such as eCRS. In this review, we discuss various mechanisms by which exogenous proteases influence eCRS and highlight the emerging role of endogenous protease inhibitors in eCRS pathogenesis.

Serum Cystatin C as a Marker for Acute Kidney Injury Evaluation in Neonates Treated with Colistin

Background Colistin is increasingly used worldwide due to the emergence of multi-drug resistant (MDR) gram negative bacilli with concerns about its side effects especially nephrotoxicity. Serum cystatin C (CysC) is an endogenous protease inhibitor produced at a constant rate by nucleated cells and used as a filtration marker of glomerular filtration rate (GFR). Aim to study the effect of colistin on serum cystatin C as a marker for acute kidney injury in neonates in relation to serum urea and serum creatinine. Patients and method 78 neonates with gestational age of at least 30 weeks after treatment with colistin or antibiotics were divided into two groups non – colistin group and colistin group 39 pateints on each group. Kidney function tests were assessed before treatment and also after treatment but together with Kidney Disease Improving Global Outcome (KDIGO) criteria & serum cystatin C. Results Serum cystatin C was found to be significantly higher in colistin group than non – colistin group. No significant differences were found between both groups regarding serum creatinine, serum urea and even KDIGO criteria of acute kidney injury in neonates. Conclusion Serum cystatin C is an early marker of acute kidney injury in neonates which may not be detected by serum urea or serum creatinine

Identification of a Tissue Inhibitor of Metalloproteinase-2: An Endogenous Inhibitor of Modori-Inducing Insoluble Metalloproteinase from Yellowtail Seriola quinqueradiata Muscle.

The existence of an endogenous protease inhibitor (EPI) was expected from the comparison of the gel properties between washed and nonwashed yellowtail surimi gels. A possible candidate, tissue inhibitor of metalloproteinase-2 (TIMP-2), was partially purified from the soluble fraction of yellowtail muscle, and an 18 kDa protein band was detected by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and western blot analysis. Its N-terminal amino acid sequence was determined as XSXSPAHPQQAF, with high homology to TIMP-2 from other fish species, suggesting that it was identified as yellowtail TIMP-2. Subsequently, full-length cDNA of two isoforms (TIMP-2a and TIMP-2b) was successfully cloned from yellowtail muscle. The N-terminal sequence of purified TIMP-2 completely corresponded to TIMP-2b. When the surimi gel quality decreased after spawning, the mRNA expression of TIMP-2b also decreased. Human TIMP-2 could inhibit autolysis of myofibrillar proteins from yellowtail muscle. Thus, TIMP-2b was considered the major EPI of the modori-inducing insoluble metalloproteinase in yellowtail muscle.

Plasma enzymatic activity, proteomics and peptidomics in COVID-19-induced sepsis: A novel approach for the analysis of hemostasis.

Introduction: Infection by SARS-CoV-2 and subsequent COVID-19 can cause viral sepsis. We investigated plasma protease activity patterns in COVID-19-induced sepsis with bacterial superinfection, as well as plasma proteomics and peptidomics in order to assess the possible implications of enhanced proteolysis on major protein systems (e.g., coagulation). Methods: Patients (=4) admitted to the intensive care units (ICUs) at the University of California, San Diego (UCSD) Medical Center with confirmed positive test for COVID-19 by real-time reverse transcription polymerase chain reaction (RT-PCR) were enrolled in a study approved by the UCSD Institutional Review Board (IRB# 190699, Protocol #20-0006). Informed consent was obtained for the collection of blood samples and de-identified use of the data. Blood samples were collected at multiple time points and analyzed to quantify a) the circulating proteome and peptidome by mass spectrometry; b) the aminopeptidase activity in plasma; and c) the endopeptidase activity in plasma using fluorogenic substrates that are cleaved by trypsin-like endopeptidases, specific clotting factors and plasmin. The one patient who died was diagnosed with bacterial superinfection on day 7 after beginning of the study. Results: Spikes in protease activity (factor VII, trypsin-like activity), and corresponding increases in the intensity of peptides derived by hydrolysis of plasma proteins, especially of fibrinogen degradation products and downregulation of endogenous protease inhibitors were detected on day 7 for the patient who died. The activity of the analyzed proteases was stable in survivors. Discussion: The combination of multiomics and enzymatic activity quantification enabled to i) hypothesize that elevated proteolysis occurs in COVID-19-induced septic shock with bacterial superinfection, and ii) provide additional insight into malfunctioning protease-mediated systems, such as hemostasis.

Endogenous secretory leukocyte protease inhibitor inhibits microbial-induced monocyte activation.

In the intestine, epithelial factors condition incoming immune cells including monocytes to adapt their threshold of activation and prevent undesired inflammation. Colonic epithelial cells express Secretory Leukocyte Protease Inhibitor (SLPI), an inhibitor of NF kappa light chain enhancer of activated B cells (NF-κB) that mediates epithelial hyporesponsiveness to microbial stimuli. Uptake of extracellular SLPI by monocytes has been proposed to inhibit monocyte activation. We questioned whether monocytes can produce SLPI and whether endogenous SLPI can inhibit monocyte activation. We demonstrate that human THP-1 monocytic cells produce SLPI and that CD68+ SLPI-producing cells can be detected in human intestinal lamina propria. Knockdown of SLPI in human THP-1 cells significantly increased NF-κB activation and subsequent C-X-C motif chemokine ligand 8 (CXCL8) and TNF-α production in response to microbial stimulation. Reconstitution of SLPI-deficient cells with either full-length SLPI or SLPI lacking its signal peptide rescued inhibition of NF-κB activation and cytokine production, demonstrating that endogenous SLPI inhibits monocytic cell activation. Unexpectedly, exogenous SLPI did not inhibit CXCL8 or TNF-α production, despite efficient uptake. Our data argue that endogenous SLPI can regulate the threshold of activation in monocytes, thereby preventing activation by commensal bacteria in mucosal tissues.

Effect of Cuban Porcine Pulmonary Surfactant (Surfacen) and rCmPI-II Protease Inhibitor on Neutrophil Elastase Activity.

In inflammatory respiratory diseases, the imbalance between proteases and endogenous protease inhibitors leads to an exacerbated activity of human neutrophil elastase (a protease that destroys the extracellular matrix and stimulates proinflammatory cytokine release). Elastase is considered a target in the search for therapeutic treatments for inflammatory respiratory diseases. Pulmonary surfactant is a promising product for this purpose, because in addition to its biophysical function, it has anti-inflammatory properties. Evaluate effect of the Cuban porcine pulmonary surfactant (Surfacen), the rCmPI-II elastase inhibitor, and the Surfacen/rCmPI-II combination on activated neutrophil elastase activity in vitro, and determine if Surfacen's interface property changes in the presence of the inhibitor. The anti-elastase effect of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination was evaluated in an in vitro model of activated neutrophils, previously purified from the blood of healthy subjects. The cells were stimulated with LPS/fMLP and were incubated with different concentrations of Surfacen, rCmPI-II and the Surfacen/rCmPI-II combination. Elastase activity was measured. The interface property was determined on a Langmuir surface balance. The new index, called the abdominal adipose deposit index, was obtained by multiplying the subcutaneous fat thickness by visceral fat thickness, both measured by ultrasound. A cutoff point was established that facilitated discernment of an unhealthy phenotype: normal weight but metabolically obese, a cardiometabolic risk factor. Surfacen at 10 mg/mL inhibited 71% of stimulated neutrophil elastase activity. rCmPI-II at 0.1 μM reduced 20% of elastase activity; at 200 μM-the maximum concentration evaluated-inhibition was 68%. Both products had a dose-dependent effect. The Surfacen/inhibitor combination (0.5 mg/mL/80 µM) did not affect the surfactant interface property or the inhibitory activity of rCmPI-II against human neutrophil elastase. Surfacen and the rCmPI-II inhibitor have an anti-elastase effect on an activated neutrophil model. rCmPI-II does not affect Surfacen's interface property and, therefore, both can be evaluated for combined use in treating inflammatory lung diseases.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection.

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad‐spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS‐CoV, MERS‐CoV, hCoV‐229E, SARS‐CoV‐2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS‐CoV‐2. Thus, AT is an endogenous inhibitor of SARS‐CoV‐2 that may be involved in COVID‐19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti‐TMPRSS2 and anti‐SARS‐CoV‐2 activity of AT, suggesting that repurposing of native and activated AT for COVID‐19 treatment should be explored.

Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

SummaryIndividuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

SerpinB7 deficiency contributes to development of psoriasis via calcium-mediated keratinocyte differentiation dysfunction

Defective execution of proteases and protease inhibitors that mediate abnormal signaling cascades is emerging as a key contributor to skin diseases, such as psoriasis. SerpinB7 is identified as a skin-specific endogenous protease inhibitor, but the role and underlying mechanism in psoriasis are poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patients and imiquimod-induced psoriatic lesions in mice. SerpinB7-/- mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to SerpinB7+/+ mice after Imiquimod treatment. Mechanistically, SerpinB7 deficiency results in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in normal human epidermal keratinocyte and mouse primary keratinocyte. Transcriptomics and proteomics results showed that the SeprinB7 deficiency affected keratinocyte differentiation and proinflammatory cytokines, possibly by affecting the calcium ion channel-related proteins. Notably, we demonstrated that SerpinB7 deficiency prevented the increase in intracellular Ca2+ influx, which was partly eliminated by the intracellular Ca2+ chelator BAPTA-AM. Our findings first described the critical role of SerpinB7 in the regulation of keratinocyte differentiation and psoriatic microenvironment mediated via keratinocytes' intracellular calcium flux, proposing a new candidate for therapeutic targets in psoriasis.

Structural and computational investigation of MMP‐3 affinity improvements with an engineered TIMP‐1 inhibitor

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that function to mediate tissue remodeling; however, they are also implicated in pathological conditions such as cancer, inflammatory and degenerative diseases. There are 23 human MMPs and significant sequence and structural homology exists among them and with other metalloproteinases. This represents a substantial challenge in developing therapeutically viable selective inhibitors due to off‐target effects. Tissue inhibitors of metalloproteinases (TIMPs) are a family of four endogenous protein protease inhibitors that regulate MMP activity. Our lab and others have utilized TIMPs as scaffolds, engineering them to selectively target specific MMPs for potential therapeutic applications. We previously employed a yeast surface display platform to identify TIMP‐1 variants possessing enhanced MMP‐3 affinity. In the current study, we used structures of MMP‐3 co‐crystallized with either WT TIMP‐1 or an engineered TIMP‐1 variant as seeds for molecular dynamics simulations. Simulations were also conducted on unbound WT and engineered TIMP‐1 to examine MMP‐independent dynamics that distinguish the engineered variant. The simulation data were analyzed with root‐mean‐square deviation/fluctuation, time‐dependent secondary structure, temporally resolved hydrogen bonding, conformational clustering, and dynamical network algorithms to discern structural and dynamical determinants of the increased affinity. Key observations that demonstrate the increased effectiveness of engineered TIMP‐1 as an MMP‐3 inhibitor were secondary structural changes in the MMP‐binding loops in proximity to mutations, attenuated global and mutation‐adjacent dynamics, decreased conformational dynamics, and increased persistence of MMP‐3‐compatible conformations. There were also indications of greater interdomain cooperativity within the engineered TIMP‐1 variant: increased interdomain hydrogen bonding, coordinated motions of the N‐terminal region with the C‐terminal domain, and increased intercommunity information flow/motion transfer. From these observations, we hypothesize the increased interdomain interactions represent a mechanism of dynamical stabilization such that mainly cooperative motion between domains presides, rather than random independent motions. Our findings suggest propagated motions between domains may influence the MMP affinity of TIMPs. Furthermore, these data suggest future strategies to design next‐generation mutant TIMP libraries by targeting regions dynamically coupled to the MMP binding loops for variation, thus exploiting natural protein dynamics to achieve desired selectivity.

Reconstructing the epidermal proteolytic cascades in health and disease.

The epidermis is the outer stratified epithelium of the skin, forming the physical barrier that is indispensable for homeostasis. Epidermal proteolysis, mainly but not exclusively executed by kallikrein-related peptidases (KLKs), is tightly regulated to ensure maintenance of physiological skin renewal and an intact skin barrier. Perturbation of epidermal proteolytic networks is implicated in a wide array of rare and common skin pathologies of diverse genetic backgrounds. Recent studies of monogenic human skin diseases and newly developed animal models have revealed new mechanisms of regulation of proteolytic pathways in epidermal physiology and in disease states. These new data have challenged some accepted views, for example the role of matriptase in epidermal desquamation, which turned out to be restricted to mouse skin. The significance of PAR2 signaling in skin inflammation should also be reconsidered in the face of recent findings. Cumulatively, recent studies necessitate a sophisticated redefinition of the proteolytic and signaling pathways that operate in human skin. We elaborate how epidermal proteolysis is finely regulated at multiple levels, and in a spatial manner that has not been taken into consideration so far, in which specific proteases are confined to distinct epidermal sublayers. Of interest, transglutaminases have emerged as regulators of epidermal proteolysis and desquamation by spatially fixing endogenous protease inhibitors, constituting regulatory factors that were not recognized before. Furthermore, new evidence suggests a link between proteolysis and lipid metabolism. By synthesis of established notions and recent discoveries, we provide an up-to-date critical evaluation and synthesis of current knowledge and the extended complexity of proteolysis regulation and signaling pathways in skin. © 2022 The Pathological Society of Great Britain and Ireland.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Aggregation of Cystatin C Changes Its Inhibitory Functions on Protease Activities and Amyloid β Fibril Formation.

Cystatin C (CST3) is an endogenous cysteine protease inhibitor, which is implicated in cerebral amyloid angiopathy (CAA). In CAA, CST3 is found to be aggregated. The purpose of this study is to investigate whether this aggregation could alter the activity of the protein relevant to the molecular pathology of CAA. A system of CST3 protein aggregation was established, and the aggregated protein was characterized. The results showed that CST3 aggregated both at 80 °C without agitation, and at 37 °C with agitation in a time-dependent manner. However, the levels of aggregation were high and appeared earlier at 80 °C. Dot-blot immunoassay for oligomers revealed that CST3 could make oligomeric aggregates at the 37 °C condition. Electron microscopy showed that CST3 could make short fibrillary aggregates at 37 °C. Cathepsin B activity assay demonstrated that aggregated CST3 inhibited the enzyme activity less efficiently at pH 5.5. At 7.4 pH, it lost the inhibitory properties almost completely. In addition, aggregated CST3 did not inhibit Aβ1-40 fibril formation, rather, it slightly increased it. CST3 immunocytochemistry showed that the protein was positive both in monomeric and aggregated CST3-treated neuronal culture. However, His6 immunocytochemistry revealed that the internalization of exogenous recombinant CST3 by an astrocytoma cell culture was higher when the protein was aggregated compared to its monomeric form. Finally, MTT cell viability assay showed that the aggregated form of CST3 was more toxic than the monomeric form. Thus, our results suggest that aggregation may result in a loss-of-function phenotype of CST3, which is toxic and responsible for cellular degeneration.

26415 Regulation of postinflammatory hyperpigmentation by niacinamide through potential modulation of the protease inhibitor, SERPINB3

Among various cutaneous disorders, acne is often accompanied by post inflammatory hyperpigmentation (PIH). Such increases in pigmentation are controlled not only by secreted factors from keratinocytes, but also from dermal fibroblasts which respond to inflammatory stimuli from the epidermis. Niacinamide is a cosmetic ingredient with proven clinical efficacy against acne-associated PIH. In this study we evaluated the role of Serpin Family B Member 3 (SERPINB3), an endogenous protease inhibitor reported to be upregulated in acne lesions as a potential regulator of the PIH response and its modulation by niacinamide.

The endogenous cellular protease inhibitor SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity.

COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown, while overexpression lead to a drastic reduction of the viral load. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.