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Joel Habener, Svetlana Mojsov, and Lotte Bjerre Knudsen have been awarded the 2024 Lasker-DeBakey Clinical Medical Research Award for their work on GLP-1 receptor agonists and their applications to obesity. GLP-1 receptor agonists have revolutionized the treatment of metabolic disorders like diabetes and obesity, and have shown potential for treating other disorders as well. These drugs have been studied in cardiovascular trials and have shown significant reductions in cardiovascular outcomes, as well as beneficial effects on blood pressure, lipoproteins, and inflammation. However, they do come with side effects such as nausea, vomiting, pancreatitis, and injection site reactions. Longer-acting forms of GLP-1 receptor agonists have been developed to improve convenience for patients. [Extracted from the article]

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

Objective • This study was carried out to evaluate the clinical efficacy of proprotein convertase chymotrypsin 9 (PCSK9) inhibitors in multi-branch lesions in coronary artery disease with substandard lipid-lowering effects. Methods • This retrospective study collected the clinical data of 100 patients with multiple coronary artery diseases admitted to our hospital between May 2020 and August 2022 for analysis. The eligible patients were assigned to either a PCSK9 inhibitor group or a control group at a ratio of 1:1 by their dosing regimens, with 50 cases in each group. Outcome measures for the clinical efficacy of PCSK9 inhibitors included lipid levels, low-density lipoprotein cholesterol (LDL-C) changes, serum concentrations of coronary artery disease-related inflammatory factors, improvement of angina questionnaire scores, adverse reactions, and major cardiovascular adverse events. Results • PCSK9 inhibitors resulted in significantly lower serum concentrations of total cholesterol (TC), LDL-C, and ApoB and higher high-density lipoprotein cholesterol (HDL-C) levels versus conventional lipid-lowering medication (P < .05). The two arms exhibited similar serum concentrations of triglyceride (TG) and ApoA1 after treatment (P > .05). With LDL-C<1.4 mmol/L as the cut-off for desirable blood lipid levels, 47 (94%) patients reached the standard after in the PCSK9 inhibitors group, while no eligible cases were reported in the control group (P < .05). PCSK9 inhibitors provided a marked reduction in the serum concentrations of high-sensitivity C-reactive protein in the patients. Patients had higher angina stability (AS), angina flare (AF), physical limitation (PL), and treatment satisfaction (TS) scores after PCSK9 inhibitor administration versus after conventional medication (P < .05). PCSK9 inhibitors were associated with a significantly lower incidence of adverse cardiovascular events (10%) versus conventional medication (42%) (P < .05). Conclusion • PCSK9 inhibitors significantly improve the LDL-C concentrations of patients with multiple lesions of coronary artery disease who have failed to meet lipidlowering targets, this enables physicians to more effectively manage patients’ cholesterol levels, consequently reducing their cardiovascular risk. Moreover, these inhibitors have the potential to enhance patients’ quality of life by alleviating relieve angina symptoms. These findings offer valuable insights into managing multi-branch coronary artery disease. [ABSTRACT FROM AUTHOR]

Objective(s): Stress elicits physiological and neuroendocrine responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis and lateral hypothalamus (LH). However, prolonged stress can dysregulate neuropeptide systems like orexin. This study investigated the effects of temporary and prolonged stress on HPA activity and orexin processing in the rat LH. Materials and Methods: Male Wistar rats were exposed to various stress repetitions. The stress paradigm is defined as short (acute; 1 day and mild; 3 days) and long (sub-chronic; 10 days and chronic; 21 days)-term 6 hr daily restraint stress. Plasma corticosterone (CORT) served as an index of HPA function. Expression of prepro-orexin and its processing enzymes prohormone convertases (PC) 1 and 2 was measured in LH tissues using semiquantitative RT-PCR. Results: The plasma level of CORT was elevated following mild, sub-chronic, and chronic, but not acute stress versus unstressed controls. The expression of prepro-orexin was heightened following all stress exposures. However, PC1 increased and PC2 decreased only after prolonged stress. The PC1/PC2 ratio was also selectively augmented with sub-chronic and chronic stress, implying impaired orexin maturation. Conclusion: Together, these data demonstrate that the HPA axis and lateral hypothalamic orexin system respond to stress based on stress repetition. Changes in orexin processing enzyme mRNA, exclusively after chronic stress, imply potential effects on peptide maturation, requiring confirmation of the orexin production at the protein level. [ABSTRACT FROM AUTHOR]

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein. [ABSTRACT FROM AUTHOR]

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P

BackgroundPCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9.MethodsWe obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay.ResultsMedian plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant (P=0.83 [95% CI, -6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P=0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (RS=-0.3634; P

Background: Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2). Results: Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule. Conclusions: In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). Methods: The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. Results: Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs' inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. Conclusions: PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC. [ABSTRACT FROM AUTHOR]

The burden of atherosclerotic cardiovascular disease (ASCVD) persists globally, demanding innovative therapeutic strategies. This manuscript provides an expert opinion on the significance of managing low-density lipoprotein cholesterol in ASCVD prevention and introduces inclisiran, a novel small interfering RNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). This work delves into the intricate mechanism of inclisiran, highlighting its unique approach of hepatic intracellular PCSK9 inhibition, its precision and low off-target effects risk. Pharmacodynamic and pharmacokinetic distinctions from PCSK9 monoclonal antibodies are explored, underlining inclisiran's efficiency, extended duration, and clearance. Clinical trials, including pivotal phase-III placebo-controlled studies (ORION-9, −10, −11), the open-label ORION-3 and pooled safety analysis of these trails including the open-label phase of ORION-8, as well as real-word data are discussed to provide a comprehensive evaluation of inclisiran's efficacy and safety. Inclisiran stands as a first-in-class breakthrough in lipid-lowering therapies, showing potential in alleviating the global burden of ASCVD and is supported by multiple global regulatory approvals. To optimize inclisiran's utilization and comprehend its long-term effects, future directions include pediatric studies, cardiovascular outcome trials, and extended-duration investigations. Overall, inclisiran emerges as a precise and effective therapeutic option, offering significant promise for preserving cardiovascular health. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a posttranslational regulator of the LDL receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-Seq-based variant discovery, we identified a possible psoriasis-susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C > T). This finding was verified in independently acquired genomic and RNA-Seq data sets. Single-cell RNA-Seq (scRNA-Seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-Seq and IHC demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression, including IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C > T was associated with lower PCSK9 expression and higher IL36G expression, when compared with heterozygous skin or cell lines. Together, these results support PCSK9 as a psoriasis-susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.

The cleavage of viral surface proteins by furin is associated with some viruses' high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach. [ABSTRACT FROM AUTHOR]

Objectives: Furin (Proprotein Convertase Subtilisin/Kexin Type 3, PCSK3) is a proprotein convertase involved in the processing of precursor proteins. Furin substrates play significant roles in the initiation and progression of atherosclerosis, which is the primary cause of coronary artery disease (CAD). This study aimed to investigate the serum furin concentrations in stable CAD patients and their relationship with disease severity. Methods: The study included 81 stable CAD patients and 50 subjects without coronary artery lesions. Coronary angiography was performed via the percutaneous femoral artery approach, and the severity of CAD was assessed using the Gensini score. Serum furin concentrations were measured using an enzyme-linked immunosorbent assay. Results: Serum furin concentrations were significantly higher in CAD patients compared to CAD-negative subjects (p=0.0001). The serum furin levels of mild, moderate, and severe CAD patients were 1.53 ng/mL, 2.01 ng/mL, and 3.03 ng/mL, respectively, which were significantly different from CAD-negative subjects (p=0.018, p=0.002, and p=0.0001, respectively). Furin levels were found to be an independent predictor of CAD and exhibited potential diagnostic value for CAD and severe CAD. Conclusion: The study concluded that serum furin concentrations could be considered a new risk factor for CAD, in addition to well-known biomarkers. [ABSTRACT FROM AUTHOR]

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid-phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with Nε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by ~50% towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin-kexins (PCSKs) type proteases, and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80% in a concentration-dependent manner. Here, we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing, are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE modification relevant for the etiopathogenesis of diabetes and the development of obesity. [ABSTRACT FROM AUTHOR]

Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a β-cell-specific Cpe knockout mouse model. Increases in dibasic residue–containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms. [ABSTRACT FROM AUTHOR]

The emerging viruses SARS‐CoV‐2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS‐CoV‐2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme‐1/site‐1 protease (SKI‐1/S1P), respectively. We report improved luciferase‐based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high‐throughput manner. We identified 23 FDA‐approved small molecules, among them monensin which displayed broad activity against furin and SKI‐1/S1P. Monensin inhibited arenaviruses and SARS‐CoV‐2 in a dose‐dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS‐CoV‐2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS‐CoV‐2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad‐spectrum antivirals with therapeutic potential to combat current and future emerging viruses. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) play important roles in regulating several human diseases, like cancer, neurodegenerative disorders, and metabolic disorders. Investigating PTMs' contribution to protein functions is critical for modern biology and medicine. Proprotein convertases (PCs) are irreversible post-translational modifiers that have been extensively studied and are considered as key targets for novel therapeutics. They cleave proteins at specific sites causing conformational changes affecting their functions. Furin is considered as a PC model in regulating growth factors and is involved in regulating many pro-proteins. The mammalian target of the rapamycin (mTOR) signaling pathway is another key player in regulating cellular processes and its dysregulation is linked to several diseases including type 2 diabetes (T2D). The role of furin in the context of diabetes has been rarely explored and is currently lacking. Moreover, furin variants have altered activity that could have implications on overall health. In this review, we aim to highlight the role of furin in T2D in relation to mTOR signaling. We will also address furin genetic variants and their potential effect on T2D and β-cell functions. Understanding the role of furin in prediabetes and dissecting it from other confounding factors like obesity is crucial for future therapeutic interventions in metabolic disorders. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic LDL receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL-cholesterol (LDL-C) levels and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3'-UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3'-UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression, and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p levels. In mice, hepatic miR-483 overexpression increased LDLR levels by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3'-UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3'-UTR (ΔBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.

Clinical management of cystic fibrosis (CF) has been greatly improved by the development of small molecule modulators of the CF transmembrane conductance regulator (CFTR). These drugs help to address some of the basic genetic defects of CFTR; however, no suitable CFTR modulators exist for 10% of people with CF (PWCF). An alternative, mutation-agnostic therapeutic approach is therefore still required. In CF airways, elevated levels of the proprotein convertase furin contribute to the dysregulation of key processes that drive disease pathogenesis. Furin plays a critical role in the proteolytic activation of the epithelial sodium channel; hyperactivity of which causes airways dehydration and loss of effective mucociliary clearance. Furin is also responsible for the processing of transforming growth factor-ß, which is increased in bronchoalveolar lavage fluid from PWCF and is associated with neutrophilic inflammation and reduced pulmonary function. Pathogenic substrates of furin include Pseudomonas exotoxin A, a major toxic product associated with Pseudomonas aeruginosa infection and the spike glycoprotein of severe acute respiratory syndrome coronavirus 2, the causative pathogen for coronavirus disease 2019. In this review we discuss the importance of furin substrates in the progression of CF airways disease and highlight selective furin inhibition as a therapeutic strategy to provide clinical benefit to all PWCF. [ABSTRACT FROM AUTHOR]

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types (μ, δ, or κ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides.

Hepatocytes function largely through the secretion of proteins that regulate cell proliferation, metabolism, and intercellular communications. During the progression of hepatocellular carcinoma (HCC), the hepatocyte secretome changes dynamically as both a consequence and a causative factor in tumorigenesis, although the full scope of secreted protein function in this process remains unclear. Here, we show that the secreted pseudo serine protease PRSS35 functions as a tumor suppressor in HCC. Mechanistically, we demonstrate that active PRSS35 is processed via cleavage by proprotein convertases. Active PRSS35 then suppresses protein levels of CXCL2 through targeted cleavage of tandem lysine (KK) recognition motif. Consequently, CXCL2 degradation attenuates neutrophil recruitment to tumors and formation of neutrophil extracellular traps, ultimately suppressing HCC progression. These findings expand our understanding of the hepatocyte secretome's role in cancer development while providing a basis for the clinical translation of PRRS35 as a therapeutic target or diagnostic biomarker. The secretome of hepatocytes and hepatocellular carcinoma (HCC) cells can contribute to cancer progression. Here the authors show that PRSS35 inhibits HCC progression through proteolytic depletion of CXCL2 and subsequently decreased neutrophil recruitment to tumours. [ABSTRACT FROM AUTHOR]

The cleavage of viral surface proteins by furin is associated with some viruses’ high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach.

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2′. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2′ formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2′ into a new C-terminal fragment (S2″), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2″ formation abolished S2′ and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2″ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms. [ABSTRACT FROM AUTHOR]

Insulin secretion is critical for glucose homeostasis, and increased levels of the precursor proinsulin relative to insulin indicate pancreatic islet beta-cell stress and insufficient insulin secretory capacity in the setting of insulin resistance. We conducted meta-analyses of genome-wide association results for fasting proinsulin from 16 European-ancestry studies in 45,861 individuals. We found 36 independent signals at 30 loci (p value < 5 × 10−8), which validated 12 previously reported loci for proinsulin and ten additional loci previously identified for another glycemic trait. Half of the alleles associated with higher proinsulin showed higher rather than lower effects on glucose levels, corresponding to different mechanisms. Proinsulin loci included genes that affect prohormone convertases, beta-cell dysfunction, vesicle trafficking, beta-cell transcriptional regulation, and lysosomes/autophagy processes. We colocalized 11 proinsulin signals with islet expression quantitative trait locus (eQTL) data, suggesting candidate genes, including ARSG , WIPI1 , SLC7A14 , and SIX3. The NKX6-3/ANK1 proinsulin signal colocalized with a T2D signal and an adipose ANK1 eQTL signal but not the islet NKX6-3 eQTL. Signals were enriched for islet enhancers, and we showed a plausible islet regulatory mechanism for the lead signal in the MADD locus. These results show how detailed genetic studies of an intermediate phenotype can elucidate mechanisms that may predispose one to disease. Broadaway et al. describe a genome-wide association meta-analysis in which they identify 36 proinsulin signals. Identification and integration of the proinsulin signals with glycemic traits, expression data in trait-relevant tissues, and functional follow-up provide hypotheses about potential mechanistic pathways for T2D loci. [ABSTRACT FROM AUTHOR]

Protein convertases (PCs) play a significant role in post-translational procedures by transforming inactive precursor proteins into their active forms. The role of PCs is crucial for cellular homeostasis because they are involved in cell signaling. They have also been described in many diseases such as Alzheimer's and cancer. Cancer cells are secretory cells that send signals to the tumor microenvironment (TME), remodeling the surrounding space for their own benefits. One of the most important components of the TME is the immune system of the tumor. In this review, we describe recent discoveries that link PCs to the immune escape of tumors. Among PCs, many findings have determined the role of Furin (PC3) as a paramount enzyme causing the TME to induce tumor immune evasion. The overexpression of various cytokines and proteins, for instance, IL10 and TGF-B, moves the TME towards the presence of Tregs and, consequently, immune tolerance. Furthermore, Furin is implicated in the regulation of macrophage activity that contributes to the increased impairment of DCs (dendritic cells) and T effector cells. Moreover, Furin interferes in the MHC Class_1 proteolytic cleavage in the trans-Golgi network. In tumors, the T cytotoxic lymphocytes (CTLs) response is impeded by the PD1 receptor (PD1-R) located on CTLs and its ligand, PDL1, located on cancer cells. The inhibition of Furin is a subtle means of enhancing the antitumor response by repressing PD-1 expression in tumors or macrophage cells. The impacts of other PCs in tumor immune escape have not yet been clarified to the extent that Furin has. Accordingly, the influence of other types of PCs in tumor immune escape is a promising topic for further consideration. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) in response to elevated endoplasmic reticulum (ER) stress. Whereas the onset of simple steatosis requires elevated de novo lipogenesis, progression to NASH is triggered by accumulation of hepatocyte-free cholesterol. We now show that caspase-2, whose expression is ER-stress inducible and elevated in human and mouse NASH, controls the buildup of hepatic-free cholesterol and triglycerides by activating sterol regulatory element-binding proteins (SREBP) in a manner refractory to feedback inhibition. Caspase-2 colocalizes with site 1 protease (S1P) and cleaves it to generate a soluble active fragment that initiates SCAP-independent SREBP1/2 activation in the ER. Caspase-2 ablation or pharmacological inhibition prevents diet-induced steatosis and NASH progression in ER-stress-prone mice. Caspase-2 inhibition offers a specific and effective strategy for preventing or treating stress-driven fatty liver diseases, whereas caspase-2-generated S1P proteolytic fragments, which enter the secretory pathway, are potential NASH biomarkers.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible β cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the β cell GLP-1R in islet function. Our data show that loss of β cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a β cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the β cell GLP-1R as a regulator of α cell proglucagon processing.

Recent studies identified a missense mutation in the gene coding for G protein-coupled receptor kinase 6 (GRK6) that segregates with type 2 diabetes (T2D). To better understand how GRK6 might be involved in T2D, we used pharmacological inhibition and genetic knockdown in the mouse β-cell line, MIN6, to determine whether GRK6 regulates insulin dynamics. We show inhibition of GRK5 and GRK6 increased insulin secretion but reduced insulin processing while GRK6 knockdown revealed these same processing defects with reduced levels of cellular insulin. GRK6 knockdown cells also had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing. In support of these findings, we demonstrate GRK6 rescue experiments in knockdown cells restored insulin secretion after glucose treatment. The altered insulin profile appears to be caused by changes in the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as GRK6 knockdown resulted in significantly reduced convertase expression and activity. To identify how the GRK6-P384S mutation found in T2D patients might affect insulin processing, we performed biochemical and cell biological assays to study the properties of the mutant. We found that while GRK6-P384S was more active than WT GRK6, it displayed a cytosolic distribution in cells compared to the normal plasma membrane localization of GRK6. Additionally, GRK6 overexpression in MIN6 cells enhanced proinsulin processing, while GRK6-P384S expression had little effect. Taken together, our data show that GRK6 regulates insulin processing and secretion in a glucose-dependent manner and provide a foundation for understanding the contribution of GRK6 to T2D. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexins (PCSKs) constitute a family of nine related proteases: PCSK1-7, MBTPS1, and PCSK9. Apart from PCSK9, little is known about PCSKs in cardiovascular disease. Here, we aimed to investigate the expression landscape and druggability potential of the entire PCSK family for CVD. We applied an integrative approach, combining genetic, transcriptomic and proteomic data from three vascular biobanks comprising carotid atherosclerosis, thoracic and abdominal aneurysms, with patient clinical parameters and immunohistochemistry of vascular biopsies. Apart from PCSK4, all PCSK family members lie in genetic regions containing variants associated with human cardiovascular traits. Transcriptomic analyses revealed that FURIN, PCSK5, MBTPS1 were downregulated, while PCSK6/7 were upregulated in plaques vs. control arteries. In abdominal aneurysms, FURIN, PCSK5, PCSK7, MBTPS1 were downregulated, while PCSK6 was enriched in diseased media. In thoracic aneurysms, only FURIN was significantly upregulated. Network analyses of the upstream and downstream pathways related to PCSKs were performed on the omics data from vascular biopsies, revealing mechanistic relationships between this protein family and disease. Cell type correlation analyses and immunohistochemistry showed that PCSK transcripts and protein levels parallel each other, except for PCSK9 where transcript was not detected, while protein was abundant in vascular biopsies. Correlations to clinical parameters revealed a positive association between FURIN plaque levels and serum LDL, while PCSK6 was negatively associated with Hb. PCSK5/6/7 were all positively associated with adverse cardiovascular events. Our results show that PCSK6 is abundant in plaques and abdominal aneurysms, while FURIN upregulation is characteristic for thoracic aneurysms. PCSK9 protein, but not the transcript, was present in vascular lesions, suggesting its accumulation from circulation. Integrating our results lead to the development of a novel 'molecular' 5D framework. Here, we conducted the first integrative study of the proprotein convertase family in this context. Our results using this translational pipeline, revealed primarily PCSK6, followed by PCSK5, PCSK7 and FURIN, as proprotein convertases with the highest novel therapeutic potential. [ABSTRACT FROM AUTHOR]

Purpose: We identified 2 individuals with de novo variants in SREBF2 that disrupt a conserved site 1 protease (S1P) cleavage motif required for processing SREBP2 into its mature transcription factor. These individuals exhibit complex phenotypic manifestations that partially overlap with sterol regulatory element binding proteins (SREBP) pathway-related disease phenotypes, but SREBF2-related disease has not been previously reported. Thus, we set out to assess the effects of SREBF2 variants on SREBP pathway activation., Methods: We undertook ultrastructure and gene expression analyses using fibroblasts from an affected individual and utilized a fly model of lipid droplet (LD) formation to investigate the consequences of SREBF2 variants on SREBP pathway function., Results: We observed reduced LD formation, endoplasmic reticulum expansion, accumulation of aberrant lysosomes, and deficits in SREBP2 target gene expression in fibroblasts from an affected individual, indicating that the SREBF2 variant inhibits SREBP pathway activation. Using our fly model, we discovered that SREBF2 variants fail to induce LD production and act in a dominant-negative manner, which can be rescued by overexpression of S1P., Conclusion: Taken together, these data reveal a mechanism by which SREBF2 pathogenic variants that disrupt the S1P cleavage motif cause disease via dominant-negative antagonism of S1P, limiting the cleavage of S1P targets, including SREBP1 and SREBP2., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Proteins destined for secretion - after removal of the signal sequence - often undergo further proteolytic processing by proprotein convertases (PCs). Prohormones are typically processed in the regulated secretory pathway, while most plasma proteins travel though the constitutive pathway. The complement system is a major proteolytic cascade in the blood, serving as a first line of defense against microbes and also contributing to the immune homeostasis. Several complement components, namely C3, C4, C5 and factor I (FI), are multi-chain proteins that are apparently processed by PCs intracellularly. Cleavage occurs at consecutive basic residues and probably also involves the action of carboxypeptidases. The most likely candidate for the intracellular processing of complement proteins is furin, however, because of the overlapping specificities of basic amino acid residue-specific proprotein convertases, other PCs might be involved. To our surprise, we have recently discovered that processing of another complement protein, mannan-binding lectin-associated serine protease-3 (MASP-3) occurs in the blood by PCSK6 (PACE4). A similar mechanism had been described for the membrane protease corin, which is also activated extracellularly by PCSK6. In this review we intend to point out that the proper functioning of the complement system intimately depends on the action of proprotein convertases. In addition to the non-enzymatic components (C3, C4, C5), two constitutively active complement proteases are directly activated by PCs either intracellularly (FI), or extracellularly (MASP-3), moreover indirectly, through the constitutive activation of pro-factor D by MASP-3, the activity of the alternative pathway also depends on a PC present in the blood. [ABSTRACT FROM AUTHOR]

Inflammation and atherogenic dyslipidaemia are often observed in skin diseases and represent an increased risk of cardiovascular disorders. Proprotein convertase subtilisin/kexin type 9 plays an important role in the regulation of serum low-density lipoprotein cholesterol levels. Its biological role, however, seems to go much beyond the regulation of cholesterol metabolism. The article presents potential pathophysiological links between inflammatory process and lipid disorders based on the example of psoriasis and systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3′-untranslated region (3′-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in PCSK7 3′-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on PCSK7 expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are potent medications in the toolkit for treatment of atherosclerotic cardiovascular disease. These agents have been well studied in clinical trials supporting their efficacy in dramatically reducing low-density lipoprotein cholesterol (LDL-C) and impact on cardiovascular outcomes. Since the approval of commercial use for PCSK9 inhibitors in 2015, we have also gained significant experience in the use of these therapeutics in the real-world setting. In this article, we review current guideline recommendations, clinical trial evidence on efficacy and safety as well as data on cost-effectiveness, prescription and adherence. We focus primarily on the monoclonal antibody class of PCSK9 inhibitors in this review while also touching on other types of therapeutics that are under development. [ABSTRACT FROM AUTHOR]