Protease Cathepsin Research Articles

Thioxothiazolidin derivative, 4-OST, inhibits melanogenesis by enhancing the specific recruitment of tyrosinase-containing vesicles to lysosome.

Tyrosinase catalyzes the rate-limiting step in melanin synthesis. Melanin is synthesized from l-tyrosin in the melanosomes, where tyrosinase and other melanogenic factors are recruited via the vesicle transport system. Genetic and biochemical approaches have revealed a correlation between impairments in the vesicle transport system and albinism. However, the specificity of the individual transport systems for the corresponding melanogenic factors has not been well elucidated yet. Here, we report that the thioxothiazolidin derivative, 4-OST (4-[(5E)-5-[(4-fluorophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-4-azatricyclo [5.2.1.02 ,6]dec-8-ene-3,5-dione: CAS RN. 477766-87-3) strongly inhibited melanogenesis in mouse melanoma B16F10 cells. 4-OST reduces tyrosinase protein levels without affecting its messenger RNA levels or enzymatic activity. Although a reduction in tyrosinase protein level was observed in the presence of a protein synthesis inhibitor, the reduction may be coupled with protein synthesis. Similarly, GIF-2202 (a derivative of 4-OST) lowers tyrosinase protein levels without affecting the levels of another melanogenic enzyme, tyrosinase-related protein 1 (TYRP1) level. The reduction in tyrosinase protein level is associated with an increase in the levels of the lysosomal proteinase cathepsin S. Chloroquine, a lysosome inhibitor, restored the tyrosinase protein level downregulated by GIF-2202, although no effects of other inhibitors (against proteasome, autophagy, or exocytosis) were observed. In addition, GIF-2202 segregated the immunofluorescence signals of tyrosinase from those of TYRP1. Chloroquine treatment resulted in co-localization of tyrosinase and cathepsin S signals near the perinuclear region, suggesting that 4-OST and GIF-2202 may alter the destination of the tyrosinase vesicle from the melanosome to the lysosome. 4-OST and GIF-2202 can be new tools for studying the tyrosinase-specific vesicle transport system.

The Role of Lysosomes in the Cancer Progression: Focus on the Extracellular Matrix Degradation

Lysosomes are multifunctional cell organelles that not only provide degradation of macromolecules by lumenal acid hydrolases, but also contribute in the regulation of cell metabolism, ion homeostasis maintenance, and programmed cell death induction. The study of this compartment in various pathological conditions, including the oncological diseases by different origins is of particular interest. This article discusses the lysosome involvement in the process of malignant cell transformation, as well as the role of these organelles in the tumor cell metastasis mediated by proteolytic cleavage of the extracellular matrix components. Metabolic and morphological changes of cells during oncogenesis lead to pHdependent redistribution of lysosomes within the cell, accompanied by the secretion of lysosomal proteases cathepsins into the extracellular space. Cysteine, serine, and arginine cathepsins released by both tumor cells and tumor-associated cells catalyze the cleavage of various components of the extracellular matrix and basement membrane, or proteolytically activate other enzymes also involved in this process. In this instance, lysosomal proteases directly affect the ability of cells to invade and the metastatic potential of tumor formation. In addition, it was demonstrated the prognostic importance of some cathepsins (especially cathepsins B, K, and D) the number and activity of which in the tumor tissue and its microenvironment are associated with malignancy of the formation, as well as with a poor prognosis for patient survival and with the possibility of recurrences.

Cathepsin C promotes breast cancer lung metastasis by modulating neutrophil infiltration and neutrophil extracellular trap formation.

Lung metastasis is the major cause of breast cancer-related mortality. The neutrophil-associated inflammatory microenvironment aids tumor cells in metastatic colonization in lungs. Here, we show that tumor-secreted protease cathepsin C (CTSC) promotes breast-to-lung metastasis by regulating recruitment of neutrophils and formation of neutrophil extracellular traps (NETs). CTSC enzymatically activates neutrophil membrane-bound proteinase 3 (PR3) to facilitate interleukin-1β (IL-1β) processing and nuclear factor κB activation, thus upregulating IL-6 and CCL3 for neutrophil recruitment. In addition, the CTSC-PR3-IL-1β axis induces neutrophil reactive oxygen species production and formation of NETs, which degrade thrombospondin-1 and support metastatic growth of cancer cells in the lungs. CTSC expression and secretion are associated with NET formation and lung metastasis in human breast tumors. Importantly, targeting CTSC with compound AZD7986 effectively suppresses lung metastasis of breast cancer in a mouse model. Overall, our findings reveal a mechanism of how tumor cells regulate neutrophils in metastatic niches and support CTSC-targeting approaches for cancer treatment.

Mature viral cathepsin is required for release of viral occlusion bodies from Autographa californica multiple nucleopolyhedrovirus-infected cells

Baculovirus-infected larvae release progeny viral occlusion bodies (OBs) to enable cyclical virus transmission to new hosts. The alphabaculovirus chitinase and cathepsin enzymes cause terminal liquefaction of host insect cadavers, aiding OB dispersal. The mechanism of cell lysis required to release the OBs is unclear but here we show Autographa californica multiple nucleopolyhedrovirus cathepsin protease activity is required for efficient release of the host tissue-degrading chitinase and cathepsin enzymes and critical for release of progeny OBs from virus-infected cells. Comparisons between viruses containing or lacking cathepsin indicate that cathepsin was necessary for OB release into cultured cell media or hemolymph of insects. In addition, pharmacological inhibition of cysteine protease activity in cells during infection blocked maturation of active cathepsin and OB release from infected cells. Together, these results suggest an important link between baculovirus-induced cell lysis, the concomitant maturation of cathepsin, and cellular release of chitinase, cathepsin and progeny OBs from cells.

A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments.Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/-knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays.Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion.Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

To study the molecular interplay between TLRs and complement representing ancient danger-sensing mechanisms, we examined the regulation of the C3a/anaphylatoxin C3a receptor (C3aR) axis in normal human epidermal keratinocytes (NHEKs) by treatment with different TLR ligands. Protein staining followed by flow cytometry revealed highly constitutive intracellular expression levels of the C3aR in NHEKs. Stimulation with Poly I:C up-regulated C3aR mRNA and intra- and extracellular expression in NHEKs which showed functional relevance by up-regulating CXCL10 and down-regulating C3 expression in response to C3a. mRNA and protein levels of C3 and protease cathepsin L (CTSL) that can cleave C3 were up-regulated by the TLR3 ligand Poly I:C. Enhanced intracellular expression levels of the biologically active C3 fragment (C3a), in response to TLR3 stimulation were also detectable in NHEKs. Cathelicidin antimicrobial peptide LL-37 potentiated Poly I:C-induced C3aR, C3, and CTSL up-regulation. In conclusion, we point to a role of TLR3 to promote up-regulation of C3aR, C3, and CTSL expression levels and generation of C3a. Our data provide evidence that local generation and activation of complement components as described for T cells or myeloid cells represent a scenario which may take place in a similar way in NHEKs.

The forensic implications of the relationship between the proteolytic enzymes activity and the changes in NADH and FAD fluorescence intensity in skeletal muscle when determining the time of death (experimental study)

Evaluation of the relationship between the activity of proteolytic enzymes (cathepsin D and calpains) and the dynamics of the fluorescence intensity of the coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the rats' skeletal muscles in relation to the time of death. The proteolytic activity of enzymes in rat skeletal muscle was determined at the postmortem time points corresponding to the most significant changes in the dynamics of coenzymes NADH and FAD fluorescence intensity. The proteolytic enzymes activity was found to be low during the period of increasing intensity of NADH fluorescence observed within 3 hours after death. An increase in the activity of proteolytic enzymes was registered in 4.5 hours after death which corresponds to the initial point of decrease in NADH fluorescence intensity. In 24 hours post-mortem, corresponding to increased FAD fluorescence intensity a significant decrease in the activity of calpains was found. The results of the study suggest that the nature of the postmortem dynamics of the fluorescence intensity of coenzymes is largely due to the peculiarities of intracellular proteolysis. The study results suggest that the pattern of post mortem changes in coenzyme fluorescence intensity is largely attributable to the specifics of intracellular proteolysis. The relationship between coenzyme fluorescence and molecular mechanisms of cell death confirms the viability and feasibility of laser-induced spectroscopy for post-mortem changes assessment when determining the time of death.

Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L.

Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation in vivo. Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, RS1A, was stabilized against proteolysis by all five cathepsins (Cts L, Cts V, Cts K, Cts S, and Cts B) while inhibiting Cts L with >25-fold specificity against the other cathepsins. We further showed that this stabilized RS1A peptide could inhibit Cts L in human liver carcinoma lysates (IC50 = 19 μM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.

Targeting lysosomal cysteine protease cathepsin S reveals immunomodulatory therapeutic strategy for oxaliplatin-induced peripheral neuropathy

Rationale: Oxaliplatin-induced peripheral neuropathy (OIPN) is a common adverse effect that causes delayed treatment and poor prognosis among colorectal cancer (CRC) patients. However, its mechanism remains elusive, and no effective treatment is available.Methods: We employed a prospective cohort study of adult patients with pathologically confirmed stage III CRC receiving adjuvant chemotherapy with an oxaliplatin-based regimen for investigating OIPN. To further validate the clinical manifestations and identify a potential therapeutic strategy, animal models, and in vitro studies on the mechanism of OIPN were applied.Results: Our work found that (1) consistent with clinical findings, OIPN was observed in animal models. Targeting the enzymatic activity of cathepsin S (CTSS) by pharmacological blockade and gene deficiency strategy alleviates the manifestations of OIPN. (2) Oxaliplatin treatment increases CTSS expression by enhancing cytosol translocation of interferon response factor 1 (IRF1), which then facilitates STIM-dependent store-operated Ca2+ entry homeostasis. (3) The cytokine array demonstrated an increase in anti-inflammatory cytokines and suppression of proinflammatory cytokines in mice treated with RJW-58. (4) Mechanistically, inhibiting CTSS facilitated olfactory receptors transcription factor 1 release from P300/CBP binding, which enhanced binding to the interleukin-10 (IL-10) promoter region, driving IL-10 downstream signaling pathway. (5) Serum CTSS expression is increased in CRC patients with oxaliplatin-induced neurotoxicity.Conclusions: We highlighted the critical role of CTSS in OIPN, which provides a therapeutic strategy for the common adverse side effects of oxaliplatin.

Advanced Nanosystems for Clinical Translation

Advanced Nanosystems for Clinical Translation

Elevation of Serum Cystatin C Levels as a Predictor of Cardiovascular Risk in Type 2 Diabetes Mellitus Patients: A Case-control Study

Introduction: Patients with type 2 Diabetes Mellitus (DM) exhibit atherogenic lipid profile, increasing the risk for atherosclerosis, compared to people without diabetes. Cystatin C, a naturally occurring protease inhibitor has a key role in pathogenesis of atherosclerosis in type 2 DM patients by inhibiting the elastolytic activity of arterial extracellular matrix by cysteine protease cathepsins. Increased concentration of cystatin C in serum reflects a counterbalance of the damaging elastolytic activity which is involved in the pathogenesis of atherosclerosis in type 2 DM patients. Aim: To determine the serum levels of cystatin C and lipid profile in type 2 DM patients with good and poor glycaemic control and to find out the correlation between serum cystatin C with HbA1c levels and cardiovascular risk markers (non-High-Density Lipoprotein Cholesterol (HDLc), Total Cholesterol (TC)/HDLc, Low-Density Liporotein (LDLc)/HDLc). Materials and Methods: The present case-control study included 60 diabetic patients of 35-75 years of age including both the genders. They were divided into Group A (n=30) with HbA1c ≤6.5% and Group B (n=30) with HbA1c >6.5%. Blood samples were collected in fasting state and analysed for Fasting Blood Sugar (FBS), TC, HDLc, LDLc. FBS was analysed by Hexokinase method, HbA1c by turbidimetric inhibition immunoassay and cystatin C by Immunoturbidimetric method. Other parameters like Non-HDLc and cardiovascular risk ratios TC/HDLc and LDLc/HDLc were calculated. Statistical analysis was done by student’s t-test and statistical significance was established at p-value <0.0001. Pearson’s correlation coefficient was used to examine various parameters. Results: In the present study, it was found that diabetic patients with HbA1c >6.5% showed increased levels of serum cystatin C (1.20±0.73 mg/L), TC (182±46.48 mg/dL), LDLc (137±13.86 mg/dL) and low levels of HDLc (34.7±12.43 mg/dL) compared to diabetic patients with HbA1c ≤6.5%. Increase in serum cystatin C levels correlated well with the increased levels of HbA1c (r=0.92), non-HDLc (r=0.87) and cardiovascular risk ratios like TC/HDLc (r=0.95) and LDLc/HDLc (r=0.97). Conclusion: The elevated serum levels of cystatin C levels correlated well with increased HbA1c and lipid profile indicating the dyslipidemia and proatherogenic status in type 2 diabetic patients with poor glycaemic control. Thus, cystatin C can be considered as the predictive marker for cardiovascular disease in type 2 diabetic patients.

Repurposing the McoTI-II Rigid Molecular Scaffold in to Inhibitor of 'Papain Superfamily' Cysteine Proteases.

Clan C1A or ‘papain superfamily’ cysteine proteases are key players in many important physiological processes and diseases in most living systems. Novel approaches towards the development of their inhibitors can open new avenues in translational medicine. Here, we report a novel design of a re-engineered chimera inhibitor Mco-cysteine protease inhibitor (CPI) to inhibit the activity of C1A cysteine proteases. This was accomplished by grafting the cystatin first hairpin loop conserved motif (QVVAG) onto loop 1 of the ultrastable cyclic peptide scaffold McoTI-II. The recombinantly expressed Mco-CPI protein was able to bind with micromolar affinity to papain and showed remarkable thermostability owing to the formation of multi-disulphide bonds. Using an in silico approach based on homology modelling, protein–protein docking, the calculation of the free-energy of binding, the mechanism of inhibition of Mco-CPI against representative C1A cysteine proteases (papain and cathepsin L) was validated. Furthermore, molecular dynamics simulation of the Mco-CPI–papain complex validated the interaction as stable. To conclude, in this McoTI-II analogue, the specificity had been successfully redirected towards C1A cysteine proteases while retaining the moderate affinity. The outcomes of this study pave the way for further modifications of the Mco-CPI design for realizing its full potential in therapeutics. This study also demonstrates the relevance of ultrastable peptide-based scaffolds for the development of novel inhibitors via grafting.

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection.

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Cathepsin L in COVID-19: From Pharmacological Evidences to Genetics.

The coronavirus disease 2019 (COVID-19) pandemics is a challenge without precedent for the modern science. Acute Respiratory Discomfort Syndrome (ARDS) is the most common immunopathological event in SARS-CoV-2, SARS-CoV, and MERS-CoV infections. Fast lung deterioration results of cytokine storm determined by a robust immunological response leading to ARDS and multiple organ failure. Here, we show cysteine protease Cathepsin L (CatL) involvement with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 from different points of view. CatL is a lysosomal enzyme that participates in numerous physiological processes, including apoptosis, antigen processing, and extracellular matrix remodeling. CatL is implicated in pathological conditions like invasion and metastasis of tumors, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, viral infection, and other diseases. CatL expression is up-regulated during chronic inflammation and is involved in degrading extracellular matrix, an important process for SARS-CoV-2 to enter host cells. In addition, CatL is probably involved in processing SARS-CoV-2 spike protein. As its inhibition is detrimental to SARS-CoV-2 infection and possibly exit from cells during late stages of infection, CatL could have been considered a valuable therapeutic target. Therefore, we describe here some drugs already in the market with potential CatL inhibiting capacity that could be used to treat COVID-19 patients. In addition, we discuss the possible role of host genetics in the etiology and spreading of the disease.

NETs Are Double-Edged Swords with the Potential to Aggravate or Resolve Periodontal Inflammation.

Periodontitis is a general term for diseases characterised by inflammatory destruction of tooth-supporting tissues, gradual destruction of the marginal periodontal ligament and resorption of alveolar bone. Early-onset periodontitis is due to disturbed neutrophil extracellular trap (NET) formation and clearance. Indeed, mutations that inactivate the cysteine proteases cathepsin C result in the massive periodontal damage seen in patients with deficient NET formation. In contrast, exaggerated NET formation due to polymorphonuclear neutrophil (PMN) hyper-responsiveness drives the pathology of late-onset periodontitis by damaging and ulcerating the gingival epithelium and retarding epithelial healing. Despite the gingival regeneration, periodontitis progression ends with almost complete loss of the periodontal ligament and subsequent tooth loss. Thus, NETs help to maintain periodontal health, and their dysregulation, either insufficiency or surplus, causes heavy periodontal pathology and edentulism.

Cathepsin V suppresses GATA3 protein expression in luminal A breast cancer

BackgroundLysosomal cysteine protease cathepsin V has previously been shown to exhibit elevated expression in breast cancer tissue and be associated with distant metastasis. Research has also identified that cathepsin V expression is elevated in tumour tissues from numerous other malignancies, but despite this, there has been limited examination of the function of this protease in cancer. Here we investigate the role of cathepsin V in breast cancer in order to delineate the molecular mechanisms by which this protease contributes to tumourigenesis.MethodsLentiviral transductions were used to generate shRNA cell line models, with cell line validation undertaken using RQ-PCR and Western blotting. Phenotypic changes of tumour cell biology were examined using clonogenic and invasion assays. The relationship between GATA3 expression and cathepsin V was primarily analysed using Western blotting. Site-directed mutagenesis was used to generate catalytic mutant and shRNA-resistant constructs to confirm the role of cathepsin V in regulating GATA3 expression.ResultsWe have identified that elevated cathepsin V expression is associated with reduced survival in ER-positive breast cancers. Cathepsin V regulates the expression of GATA3 in ER-positive breast cancers, through promoting its degradation via the proteasome. We have determined that depletion of cathepsin V results in elevated pAkt-1 and reduced GSK-3β expression, which rescues GATA3 from proteasomal degradation.ConclusionsIn this study, we have identified that cysteine protease cathepsin V can suppress GATA3 expression in ER-positive breast cancers by facilitating its turnover via the proteasome. Therefore, targeting cathepsin V may represent a potential therapeutic strategy in ER-positive breast cancers, by restoring GATA3 protein expression, which is associated with a more favourable clinical outcome.

Cathepsin S provokes interleukin-6 (IL-6) trans-signaling through cleavage of the IL-6 receptor in vitro

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. Regenerative and anti-inflammatory activities are mediated via classic signaling, in which IL-6 binds to the membrane-bound IL-6 receptor (IL-6R). For IL-6 trans-signaling, which accounts for the pro-inflammatory properties of the cytokine, IL-6 activates its target cells via soluble forms of the IL-6R (sIL-6R). We have previously shown that the majority of sIL-6R in human serum originates from proteolytic cleavage and mapped the cleavage site of the IL-6R. The cleavage occurs between Pro-355 and Val-356, which is the same cleavage site that the metalloprotease ADAM17 uses in vitro. However, sIL-6R serum levels are unchanged in hypomorphic ADAM17ex/ex mice, making the involvement of ADAM17 questionable. In order to identify other proteases that could be relevant for sIL-6R generation in vivo, we perform a screening approach based on the known cleavage site. We identify several candidate proteases and characterize the cysteine protease cathepsin S (CTSS) in detail. We show that CTSS is able to cleave the IL-6R in vitro and that the released sIL-6R is biologically active and can induce IL-6 trans-signaling. However, CTSS does not use the Pro-355/Val-356 cleavage site, and sIL-6R serum levels are not altered in Ctss−/− mice. In conclusion, we identify a novel protease of the IL-6R that can induce IL-6 trans-signaling, but does not contribute to steady-state sIL-6R serum levels.

Loss of progranulin leads to dysregulation of innate and adaptive immune cell populations, increased susceptibility to experimental colitis, and brain infiltration of peripheral immune cells

AbstractBackgroundMutations in the progranulin gene (GRN) reduce levels of progranulin (PGRN) and granulins (GRNs) causing frontotemporal dementia (FTD), the most common form of early‐onset dementia. PGRN loss in neurons is associated with lysosome dysfunction resulting in diminished cathepsin protease activity. PGRN is also highly expressed in brain‐resident microglia and peripheral immune cells. Therefore, we asked if PGRN loss impacts immune cell profiles and communication in the gut‐brain axis during chronic systemic inflammation.MethodsWe measured lysosomal‐associated proteases in microglia and peripheral immune cells using probes for cathepsin D and pan‐cysteine cathepsins; performed multiplexed RNA & digital spatial protein profiling analyses, immunohistochemistry, and deep‐immunophenotyping by flow cytometry on brain, spleen, and peripheral blood of Grn KO and Grn‐suffficient mice aged 3‐30 months. Furthermore, we exposed mice to experimental colitis to interrogate alterations in the gut‐brain axis.ResultsHighest cathepsin D activity was found amongst Ly6C+, CD11b+ blood monocytes while pan‐cathepsin activity was highest in neutrophils and monocytes. In the spleen of aged Grn KO mice, T cell counts were reduced while Ly6C‐, CD68+ macrophages were increased compared to age‐matched Grn‐sufficient mice. In blood, aberrant T cell and monocyte activation markers in Grn KO mice were evident with minimal increases in inflammatory cytokines in plasma. Aged Grn KO mice exhibited decreased microglia (CD45int, CD11bhi) in the brain relative to Grn‐sufficient mice, with altered MHCII and CD68 expression. Additionally, CD4+ and CD8+ T cells were impacted in the brain of Grn KO mice compared to age‐matched Grn‐sufficient mice. PGRN loss was also associated with increased brain infiltration of activated peripheral monocytes and, interestingly, increased vulnerability to experimental colitis that was mitigated by loss of CCR2 function.ConclusionsThese novel findings suggest that PGRN plays key regulatory functions in innate and adaptive immune cell populations, in central‐peripheral immune cell crosstalk and traffic to the CNS, and in regulating susceptibility to immune challenges that disrupt the gut‐brain signaling axis. Analysis of peripheral immune cells and colonic tissue from patients with Grn mutations or SNPs is ongoing and may shed light on the mechanisms by which GRN haploinsufficiency causes FTD.

An atypical and functionally diverse family of Kunitz-type cysteine/serine proteinase inhibitors secreted by the helminth parasite Fasciola hepatica

Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants < 10 nM). Their unusual inhibitory properties are due to (a) Leu15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1′ subsites of the cathepsin protease, and (b) Arg19 which forms cation-π interactions with Trp291 of the S1′ subsite and electrostatic interactions with Asp125 of the S2′ subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu15 in the reactive loop with Arg15. The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.

Intravascular Molecular Imaging: Near-Infrared Fluorescence as a New Frontier.

Despite exciting advances in structural intravascular imaging [intravascular ultrasound (IVUS) and optical coherence tomography (OCT)] that have enabled partial assessment of atheroma burden and high-risk features associated with acute coronary syndromes, structural-based imaging modalities alone do not comprehensively phenotype the complex pathobiology of atherosclerosis. Near-infrared fluorescence (NIRF) is an emerging molecular intravascular imaging modality that allows for in vivo visualization of pathobiological and cellular processes at atheroma plaque level, including inflammation, oxidative stress, and abnormal endothelial permeability. Established intravascular NIRF imaging targets include macrophages, cathepsin protease activity, oxidized low-density lipoprotein and abnormal endothelial permeability. Structural and molecular intravascular imaging provide complementary information about plaque microstructure and biology. For this reason, integrated hybrid catheters that combine NIRF-IVUS or NIRF-OCT have been developed to allow co-registration of morphological and molecular processes with a single pullback, as performed for standalone IVUS or OCT. NIRF imaging is approaching application in clinical practice. This will be accelerated by the use of FDA-approved indocyanine green (ICG), which illuminates lipid- and macrophage-rich zones of permeable atheroma. The ability to comprehensively phenotype coronary pathobiology in patients will enable a deeper understanding of plaque pathobiology, improve local and patient-based risk prediction, and usher in a new era of personalized therapy.