Multiple Proteases Research Articles

Evaluating the specificity of flavivirus proteases in Aedes aegypti cells for dengue virus 2-derived cleavage sites.

Flaviviruses are a diverse group of RNA viruses known for their significant impact on human health worldwide. We generated a series of reporters that included cleavage sequences from the dengue virus type 2 polyprotein and co-transfected with plasmids encoding various flavivirus proteases into Aedes aegypti cells, followed by fluorescent imaging and western blot analysis for the determination of proteolytic cleavage. Recombinant flavivirus NS2B3 proteases from medically significant and insect-specific flaviviruses were able to process reporters encoding cleavage sequences from the dengue virus type 2 polyprotein in vitro including proteases from dengue virus types 1-4, Zika virus, yellow fever virus, Aedes flavivirus, and cell-fusing agent virus. Reporters were not cleaved when transfected cells were infected with dengue virus type 2. Endoplasmic reticulum tethered reporters were also cleaved by protease alone but not by infectious virus. These results shed light on the ability of multiple flavivirus proteases to cleave sequences derived from outside of their genome and raise new questions concerning the requirements for effective cleavage by flavivirus proteases in trans.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Protease expression in the human and rat cumulus-oocyte complex during the periovulatory period: a role in cumulus-oocyte complex migration†.

The migratory and matrix-invading capacities of the cumulus-oocyte complex have been shown to be important for the ovulatory process. In metastatic cancers, these capacities are due to increased expression of proteases, however, there is limited information on protease expression in the cumulus-oocyte complexes. The present study examined cumulus-oocyte complex expression of plasmins, matrix metalloproteases, and A Disintegrin and Metalloproteinase with Thrombospondin Motifs family members in the rat and human. In the rat, human chorionic gonadotropin (hCG) administration increased cumulus-oocyte complex expression of Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, Adamts1, and the protease inhibitors Timp1, Timp3, and Serpine1 by 8-12h. This ovulatory induction of proteases in vivo could be mimicked by forskolin and ampiregulin treatment of cultured rat cumulus-oocyte complexes with increases observed in Mmp2, Mmp13, Mmp14, Mmp16, Mmp19, Plat, and the protease inhibitors Timp1, Timp3, and Serpine1. Comparison of expression between rat cumulus-oocyte complexes and granulosa cells at the time of ovulation showed decreased Mmp9 and increased Mmp13, Mmp14, Mmp16, Adamts1, Timp1, and Timp3 expression in the cumulus-oocyte complexes. In human, comparison of expression between cumulus and granulosa cells at the time of in vitro fertilization retrieval showed decreased MMP1, MMP2, MMP9, and ADAMTS1, while expression of MMP16, TIMP1, and TIMP3 were increased. Treatment of expanding rat cumulus-oocyte complexes with a broad spectrum matrix metalloproteases inhibitor, GM6001, significantly reduced the migration of cumulus cells in vitro. These data provide evidence that multiple proteases and their inhibitors are expressed in the cumulus-oocyte complex and play an important role in imparting the migratory phenotype of the cumulus-oocyte complex at the time of ovulation. Summary Sentence Multiple proteases and their inhibitors are induced in the cumulus-oocyte complex (COC) during the periovulatory period and potentially play an important role in imparting the migratory phenotype of the COC at the time of ovulation.

Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

Streptococcus suis Deploys Multiple ATP-Dependent Proteases for Heat Stress Adaptation.

Streptococcus suis is an important zoonotic pathogen, causing cytokine storms of Streptococcal toxic shock-like syndrome amongst humans after a wound infection into the bloodstream. To overcome the challenges of fever and leukocyte recruitment, invasive S. suis must deploy multiple stress responses forming a network and utilize proteases to degrade short-lived regulatory and misfolded proteins induced by adverse stresses, thereby adapting and evading host immune responses. In this study, we found that S. suis encodes multiple ATP-dependent proteases, including single-chain FtsH and double-subunit Clp protease complexes ClpAP, ClpBP, ClpCP, and ClpXP, which were activated as the fever of infected mice in vivo. The expression of genes ftsH, clpA/B/C, and clpP, but not clpX, were significantly upregulated in S. suis in response to heat stress, while were not changed notably under the treatments with several other stresses, including oxidative, acidic, and cold stimulation. FtsH and ClpP were required for S. suis survival within host blood under heat stress in vitro and in vivo. Deletion of ftsH or clpP attenuated the tolerance of S. suis to heat, oxidative and acidic stresses, and significantly impaired the bacterial survival within macrophages. Further analysis identified that repressor CtsR directly binds and controls the clpA/B/C and clpP operons and is relieved by heat stress. In summary, the deployments of multiple ATP-dependent proteases form a flexible heat stress response network that appears to allow S. suis to fine-tune the degradation or refolding of the misfolded proteins to maintain cellular homeostasis and optimal survival during infection.

The dynamic analysis of non-targeted metabolomics and antioxidant activity of Dendrobium officinale Kimura et Migo by the synergistic fermentation of bacteria and enzymes

The fermentation metabolites of Dendrobium officinale Kimura et Migo (D. officinale) exists a crucial influence on its antioxidant activity which further affects the comprehensive utilization of D. officinale fermentation products. In this research, multiple proteases were selected for the synergistic fermentation of D. officinale with Lactobacillus plantarum and Saccharomyces cerevisiae. GC-MS-based untargeted metabolomics was employed to analyze the metabolite characteristics and predict the metabolic pathways of fermented D. officinale liquid (FDL). In vitro chemical assay and cellular oxidative stress model were employed to evaluate the antioxidant activity of FDL. The contents of phenols, flavonoids and B vitamins were significantly increased during the synergistic fermentation of bacteria and enzymes. A total of 45 key differential metabolites were identified, consisting primarily of 16 sugars, 15 organic acids and 5 polyols. Furthermore, the changes in their types and contents were shaped by 6 critical differential metabolic pathways. As fermentation progressed, the scavenging capability of FDL on free radicals was notably augmented, and it had the ability to enhance the activity of intracellular antioxidant enzymes by suppressing the generation of intracellular ROS and MDA. The synergistic fermentation of bacteria and enzymes was beneficial for D. officinale exhibited efficient antioxidant activity.

DerP1, a house dust mite protease, directly activates airway vagal afferents and induces airway hyperreactivity after chronic exposure

Airway hyperreactivity (AHR) is one of the main symptoms of allergic asthma. Current therapeutics used in the treatment of allergic asthma do not target the AHR. Airway sensory afferents have been shown to be critical in AHR, but the mechanisms remain elusive. We have previously shown that house dust mite (HDM) extract, which contains multiple different proteases, can directly activate these airway sensory afferents in the lung via PAR1 receptors. In this study we identified the most potent protease DerP1 is capable of directly and robustly activating airway sensory vagal afferents. DerP1 caused a significant release of CGRP, a neuropeptide released after nerve terminal activation, from the airways. We observed that DerP1 caused a calcium influx in 65% of TRPV1 positive lung specific neurons. We also observed that in our two-photon ex vivo imaging of the vagal ganglia of PirtCre;R26 ‐ GCaMP6s mice, DerP1 caused a robust activation of approximately 60% of naïve airway-specific C-fibers. Calcium imaging of the lung specific vagal neurons from wild type, TRPA1 KO and TRPV1 KO revealed that the DerP1 mediated activation was absent in TRPV1 KO mice neurons. We then found that mice exposed to DerP1 chronically over a period of 12 days (without an adjuvant) had significant AHR and Th2 mediated airway inflammation. Our data suggests that direct activation of airway vagal C-fiber afferents by DerP1 via TRPV1 chronically leads to airway hyperreactivity and Th2 mediated airway inflammation. Warren Alpert Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway

Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged DELE1 showed that these stresses induced the cleavage of DELE1 to DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. Surprisingly, while the mitochondrial protease OMA1 was crucial for DELE1 cleavage in HeLa cells, it was dispensable in HEK293T cells, suggesting that multiple proteases may be involved in DELE1 cleavage. In support, we identified a role for the mitochondrial protease, HtrA2, in mediating DELE1 cleavage into DELE1-VS, and showed that a Parkinson’s disease (PD)-associated HtrA2 mutant displayed reduced DELE1 processing ability, suggesting a novel mechanism linking PD pathogenesis to mitochondrial stress. Our data further suggest that DELE1 is likely cleaved into DELE1-S in the cytosol, while the DELE1-VS form might be generated during halted translocation into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies a novel role for HtrA2 in DELE1 processing.

Multiplexed detection of viral proteases through dual response peptide-assisted nanopore sensing

Owing to their central roles in viral replication, main protease (Mpro) and papain-like protease (PLpro) are promising biomarkers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the simultaneous sensing of Mpro and PLpro activities is a valuable approach for SARS-CoV-2 detection. Herein, we developed a dual response peptide probe for simultaneously monitoring the protease activities of Mpro and PLpro using a single aerolysin nanopore. The target proteases can specifically digest the probe at the corresponding cleavage sites, releasing the output peptide fragments composed of different amino acids. These fragments generated clearly distinguishable blocking currents in nanopore recording that can be assigned to each analyte, allowing accurate differentiation among multiple proteases. The approach enabled the sensitive detection of Mpro and PLpro activity without the need for any amplification or labeling. Furthermore, with excellent discrimination ability, the method can be applied to evaluate the activity of the two model proteases against a background of exhaled breath condensate. Importantly, since peptides with specific sequences serve as catalytic substrates of multiple enzymes, the proposed strategy would be applicable for accurate and multiplex quantification of different types of enzymes, which is critical for non-invasive early disease diagnosis.

An in silico scheme for optimizing the enzymatic acquisition of natural biologically active peptides based on machine learning and virtual digestion

BackgroundAs a potential natural active substance, natural biologically active peptides (NBAPs) are recently attracting increasing attention. The traditional proteolysis methods of obtaining effective NBAPs are considerably vexing, especially since multiple proteases can be used, which blocks the exploration of available NBAPs. Although the development of virtual digesting brings some degree of convenience, the activity of the obtained peptides remains unclear, which would still not allow efficient access to the NBAPs. It is necessary to develop an efficient and accurate strategy for acquiring NBAPs. ResultsA new in silico scheme named SSA-LSTM-VD, which combines a sparrow search algorithm-long short-term memory (SSA-LSTM) deep learning and virtually digested, was presented to optimize the proteolysis acquisition of NBAPs. Therein, SSA-LSTM reached the highest Efficiency value reached 98.00 % compared to traditional machine learning algorithms, and basic LSTM algorithm. SSA-LSTM was trained to predict the activity of peptides in the proteins virtually digested results, obtain the percentage of target active peptide, and select the appropriate protease for the actual experiment. As an application, SSA-LSTM was employed to predict the percentage of neuroprotective peptides in the virtual digested result of walnut protein, and trypsin was ultimately found to possess the highest value (85.29 %). The walnut protein was digested by trypsin (WPTrH) and the peptide sequence obtained was analyzed closely matches the theoretical neuroprotective peptide. More importantly, the neuroprotective effects of WPTrH had been demonstrated in nerve damage mouse models. SignificanceThe proposed SSA-LSTM-VD in this paper makes the acquisition of NBAPs efficient and accurate. The approach combines deep learning and virtually digested skillfully. Utilizing the SSA-LSTM-VD based strategy holds promise for discovering and developing peptides with neuroprotective properties or other desired biological activities.

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.

Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Extracellular adherence proteins, members of a Staphylococcal immune evasion protein family, engage multiple neutrophil serine proteases simultaneously

Extracellular adherence proteins, members of a Staphylococcal immune evasion protein family, engage multiple neutrophil serine proteases simultaneously

Recessive SERPING1 Variant Leads to Kinin–Kallikrein System Control Failure in a Consanguineous Brazilian Family with Hereditary Angioedema

Background: Hereditary angioedema (HAE) is a severe and potentially life-threatening disease. The most common forms are caused by variants in SERPING1, resulting in C1-inhibitor (C1-INH) deficiency (HAE-C1-INH). C1-INH is a serine protease inhibitor (SERPIN) that regulates multiple proteases pathways, including the kallikrein–kinin system (KKS) and its complement. In HAE-C1-INH patients, C1-INH deficiencies affect KKS control, resulting in the development of kallikrein activity in plasma and the subsequent release of bradykinin (BK). While the overwhelming majority of disease-causing SERPING1 variants are dominant, very few recessive variants have been described. We present a large Brazilian HAE-C1-INH family with a recessive form of HAE-C1-INH. Methods: Blood samples of family members were investigated for protein levels of C1-INH, C4, C1q, and C1-INH function. The SERPING1 gene was sequenced. Results: In two severely affected sisters, we identified a homozygous missense variant in SERPING1 (NM_000062.3:c.964G>A;p.Val322Met). Fourteen family members were asymptomatic heterozygous carriers of the variant. Data regarding C1-INH function in the plasma showed that homozygous p.Val322Met strongly impacts C1-INH function to inhibit C1s and kallikrein (PKa). When heterozygously expressed, it affects the C1-INH control of C1s more than that of PKa. Conclusions: These studies of the variant’s effects on the structure–function relationship reinforce prior observations suggesting that C1-INH deficiency is a conformational disease.

USP35 promotes HCC development by stabilizing ABHD17C and activating the PI3K/AKT signaling pathway

S-palmitoylation is a reversible protein lipidation that controls the subcellular localization and function of targeted proteins, including oncogenes such as N-RAS. The depalmitoylation enzyme family ABHD17s can remove the S-palmitoylation from N-RAS to facilitate cancer development. We previously showed that ABHD17C has oncogenic roles in hepatocellular carcinoma (HCC) cells, and its mRNA stability is controlled by miR-145-5p. However, it is still unclear whether ABHD17C is regulated at the post-translational level. In the present study, we identified multiple ubiquitin-specific proteases (USPs) that can stabilize ABHD17C by inhibiting the ubiquitin-proteasome-mediated degradation. Among them, USP35 is the most potent stabilizer of ABHD17C. We found a positive correlation between the elevated expression levels of USP35 and ABHD17C, together with their association with increased PI3K/AKT pathway activity in HCCs. USP35 knockdown caused decreased ABHD17C protein level, impaired PI3K/AKT pathway, reduced proliferation, cell cycle arrest, increased apoptosis, and mitigated migration and invasion. USP35 can interact with and stabilize ABHD17C by inhibiting its ubiquitination. Overexpression of ABHD17C can rescue the defects caused by USP35 knockdown in HCC cells. In support of these in vitro observations, xenograft assay data also showed that USP35 deficiency repressed HCC development in vivo, characterized by reduced proliferation and disrupted PI3K/AKT signaling. Together, these findings demonstrate that USP35 may promote HCC development by stabilization of ABHD17C and activation of the PI3K/AKT pathway.

Identification of HDAC inhibitors as neutrophil recruitment modulators in zebrafish using a chemical library screen.

Tissue injury-induced neutrophil recruitment is a prerequisite for the initiation and amplification of inflammatory responses. Although multiple proteases and post-translational modification (PTM) enzymes regulate leukocyte recruitment, an unbiased functional screen of those enzymes regulating inflammatory leukocyte recruitment has yet to be undertaken. Here, using a zebrafish tail fin amputation (TFA) model to screen a chemical library consisting of 295 compounds that target proteases and PTM enzymes, we identified multiple histone deacetylase (HDAC) inhibitors that modulate inflammatory neutrophil recruitment. AR-42, a pan-HDAC inhibitor, was shown to inhibit neutrophil recruitment in three different zebrafish sterile tissue injury models: TFA, copper-induced neuromasts damage (CIND), and mechanical otic vesicle injury (MOVI), and a sterile murine peritonitis model. RNA sequencing analysis of AR-42-treated fish embryos revealed downregulation of neutrophil-associated cytokines/chemokines, and exogenous supplementation with recombinant human IL-1β and CXCL8 partially restored the defective neutrophil recruitment in AR-42-treated MOVI model fish embryos. We thus demonstrate that AR-42 non-cell autonomously modulates neutrophil recruitment by suppressing transcriptional expression of cytokines/chemokines, thereby identifying AR-42 as a promising anti-inflammatory drug for treating sterile tissue injury-associated diseases.

Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources.

Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing, and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of the recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation followed by ion trap confirmation. This enables a detailed examination of the reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production. All mass spectrometry data files have been deposited in the MassIVE repository under the identifier MSV000091776.

Calpain as a Therapeutic Target for Hypoxic-Ischemic Encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a complex pathophysiological process with multiple links and factors. It involves the interaction of inflammation, oxidative stress, and glucose metabolism, and results in acute and even long-term brain damage and impairment of brain function. Calpain is a family of Ca2+-dependent cysteine proteases that regulate cellular function. Calpain activation is involved in cerebral ischemic injury, and this involvement is achieved by the interaction among Ca2+, substrates, organelles, and multiple proteases in the neuronal necrosis and apoptosis pathways after cerebral ischemia. Many calpain inhibitors have been developed and tested in the biochemical and biomedical fields. This study reviewed the potential role of calpain in the treatment of HIE and related mechanism, providing new insights for future research on HIE.