Targeting Viral and Cellular Cysteine Proteases for Treatment of New Variants of SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants caused the persistence of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. The viral proteases are the most attractive targets for developing antiviral drugs. In this scenario, our study explores the use of HIV-1 protease inhibitors against SARS-CoV-2. An in silico screening of a library of HIV-1 proteases identified four anti-HIV compounds able to interact with the 3CLpro of SARS-CoV-2. Thus, in vitro studies were designed to evaluate their potential antiviral effectiveness against SARS-CoV-2. We employed pseudovirus technology to simulate, in a highly safe manner, the adsorption of the alpha (α-SARS-CoV-2) and omicron (ο-SARS-CoV-2) variants of SARS-CoV-2 and study the inhibitory mechanism of the selected compounds for cell-virus interaction. The results reported a mild activity against the viral proteases 3CLpro and PLpro, but efficient inhibitory effects on the internalization of both variants mediated by cathepsin B/L. Our findings provide insights into the feasibility of using drugs exhibiting antiviral effects for other viruses against the viral and host SARS-CoV-2 proteases required for entry.

METTL3-mediated m6A modification of lncRNA TSPAN12 promotes metastasis of hepatocellular carcinoma through SENP1-depentent deSUMOylation of EIF3I.

In a previous study, we discovered that the level of lnc-TSPAN12 was significantly elevated in hepatocellular carcinoma (HCC) and correlated with a low survival rate. However, the function and mechanism of lnc-TSPAN12 in modulating epithelial-mesenchymal transition (EMT) and metastasis in HCC remains poorly understood. This study demonstrates that lnc-TSPAN12 positively influences migration, invasion, and EMT of HCC cells in vitro and promotes hepatic metastasis in vivo. The modification of N6-methyladenosine, driven by METTL3, is essential for the stability of lnc-TSPAN12, which may partially contribute to the upregulation of lnc-TSPAN12. Mechanistically, lnc-TSPAN12 exhibits direct interactions with EIF3I and SENP1, acting as a scaffold to enhance the SENP1-EIF3I interaction. As a result, the SUMOylation of EIF3I is inhibited, preventing its ubiquitin-mediated degradation. Ultimately, this activates the Wnt/ß-catenin signaling pathway, stimulating EMT and metastasis in HCC. Our findings shed light on the regulatory mechanism of lnc-TSPAN12 in HCC metastasis and identify the lnc-TSPAN12-EIF3I/SENP1 axis as a novel therapeutic target for HCC.

rgpA-Engineered/Functionalized DNA Vaccine as a Novel Prophylactic Vaccination to Prevent Porphyromonas gingivalis-Induced Periodontitis: An in Vivo Study.

BACKGROUND: Arg-gingipain A (rgpA) and Arg-gingipain B (rgpB) are crucial virulence factors associated with Porphyromonas gingivalis (P. gingivalis) and have been recognized as promising targets for antibacterial vaccines. Although vaccines containing rgpA have shown efficacy, the incorporation of rgpB, which lacks the haemagglutinin adhesin (HA) domain, diminishes the vaccine's effectiveness. This study aims to assess the immunogenicity of the functional HA domain of rgpA in mouse periodontitis models. METHODS: A total of 24 mice were randomly divided into four groups, each receiving different immune injections: group A received phosphate-buffered saline (PBS) as an empty control; group B received pVAX1 as a negative control (NC); group C received pVAX1-HA; and group D received pVAX1-rgpA. The mice were subjected to intramuscular injections every two weeks for a total of three administrations. Prior to each immunization, blood samples were collected for antibody detection under isoflurane anesthesia. Following the final immunization, periodontitis was induced two weeks later by using sutures soaked in a P. gingivalis solution. The mice were euthanized after an additional two-week period. To assess the safety of the procedure, major organs were examined through hematoxylin-eosin (HE) staining. Subsequently, the levels of IgG, IgG1, and IgG2a in the serum were quantified via enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of inflammatory factors in the gingiva, including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-α), was determined using quantitative real-time reverse transcript PCR (qRT-PCR). The extent of bone loss in periodontal tissues was evaluated using micro-computed tomography (micro-CT) and HE staining. RESULTS: HE staining of the organs confirmed the absence of vaccine-induced toxicity in vivo. After the second immunization, both the rgpA and HA groups displayed significantly higher specific IgG titers in comparison to the NC and PBS groups (p < 0.05). Furthermore, the rgpA and HA groups exhibited a noteworthy predominance of IgG1 antibodies after three immunization doses, while there was a noticeable reduction in IgG2a levels observed following ligation with P. gingivalis sutures, as opposed to the NC and PBS groups (p < 0.05). Additionally, both the HA and rgpA groups showed a significant decrease in the expression of inflammatory factors such as IL-6, IL-1ß, and TNF-α, as well as a reduction in bone loss around periodontitis-affected teeth, when compared to the NC and PBS groups (p < 0.05). CONCLUSIONS: The results of this study demonstrate that the rgpA-engineered/functionalized HA gene vaccine is capable of eliciting a potent prophylactic immune response against P. gingivalis-induced periodontitis, effectively serving as an immunogenic and protective agent in vivo.

SENP3 mediates the activation of the Wnt/ß-catenin signaling pathway to accelerate the growth and metastasis of oesophagal squamous cell carcinoma in mice.

As a common malignant tumor, esophageal squamous cell carcinoma (ESCC) is occasionally seen in clinical practice. This type of disease has low incidence rate and mortality. The post-translational modification of small ubiquitin like modifiers (SUMO) can play a crucial role in regulating protein function, and can significantly impact the occurrence and development of diseases. SUMO-specific peptidase (SENP) affects cell activity by regulating the biological function of SUMO. SENP3 belongs to the SENP family, and available data indicate that many malignancies are associated with SENPs, it is currently unclear its role in ESCC. This study indicates that there is a high level of SENP3 expression in ESCC tumor cells. If the expression level of this gene is high, it can have a significant impact on ESCC cell lines and affect physiological activities such as invasion of KYSE170 cells. If the gene is knocked out, this situation will not occur. There is also research data indicating that this gene can effectively activate related signaling pathways, thereby promoting the physiological activities of malignant tumor cells. In a nude mouse xenograft tumor model, KYSE170 cells with SENP3 expression knockdown induced a smaller volume and weight of tumor tissue. Therefore, it can be clearly stated that SENP3 can enable Wnt/ ß- The catenin signaling pathway is stimulated, which in turn affects the physiological activities of ESCC cells, including the invasion process. The results of this article lay the foundation for clinical staff to carry out clinical management.

SENP1 inhibits ferroptosis and promotes head and neck squamous cell carcinoma by regulating ACSL4 protein stability via SUMO1.

Head and neck squamous cell carcinoma (HNSCC) is currently one of the most common malignancies with a poor prognosis worldwide. Meanwhile, small ubiquitin­like modifier (SUMO) specific peptidase 1 (SENP1) was associated with ferroptosis. However, the specific functions and underlying mechanisms of action of SENP1 in ferroptosis and tumor progression of HNSCC remain to be established. The findings of the present study implicated a novel ferroptosis pathway in the initiation and progression of HNSCC, providing new functional targets to guide future therapy. In the present study, The Cancer Genome Atlas database was employed to establish a gene model related to ferroptosis and verified SENP1 as a key gene via transcriptome sequencing. Expression of SENP1 in HNSCC tissue and CAL­27 cells was detected based on reverse transcription­quantitative PCR and western blot analysis. Proliferation and migration abilities of cells were determined using Cell Counting Kit­8, wound healing and Transwell experiments. Expression levels of iron, glutathione (GSH) and lipid peroxidation end­product malondialdehyde (MDA) under conditions of silencing of SENP1 with shRNA lentivirus were assayed. Additionally, the relationship between SENP1 and long­chain acyl­coenzyme A synthase 4 (ACSL4) was validated with the aid of immunoblotting and co­immunoprecipitation (co­IP). Finally, the influence of shSENP1 on the expression of key ferroptosis proteins, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11, was evaluated via western blotting. It was revealed that SENP1 was significantly overexpressed in HNSCC and associated with low patient survival. Silencing of SENP1 led to significant suppression of cell proliferation, migration and invasion, increase in the contents of iron ions and MDA and decline in GSH levels in HNSCC cells, thereby enhancing ferroptosis and inhibiting disease progression. Conversely, overexpression of SENP1 suppressed ferroptosis and promoted progression of HNSCC. Co­IP and western blot analyses revealed a SUMOylation link between SENP1 and ACSL4. SENP1 reduced the stability of ACSL4 protein through deSUMOylation, leading to inhibition of ferroptosis. SENP1 silencing further inhibited the expression of the key iron death protein, GPX4, to regulate ferroptosis. Taken together, SENP1 deficiency promoted ferroptosis and inhibited tumor progression through reduction of SUMOylation of ACSL4 in HNSCC. The collective results of the present study supported the utility of SENP1 as an effective predictive biomarker for targeted treatment of HNSCC.

SENP3 Promotes Mantle Cell Lymphoma Development through Regulating Wnt10a Expression.

OBJECTIVE: SUMO-specific protease 3 (SENP3), a member of the SUMO-specific protease family, reverses the SUMOylation of SUMO-2/3 conjugates. Dysregulation of SENP3 has been proven to be involved in the development of various tumors. However, its role in mantle cell lymphoma (MCL), a highly aggressive lymphoma, remains unclear. This study was aimed to elucidate the effect of SENP3 in MCL. METHODS: The expression of SENP3 in MCL cells and tissue samples was detected by RT-qPCR, Western blotting or immunohistochemistry. MCL cells with stable SENP3 knockdown were constructed using short hairpin RNAs. Cell proliferation was assessed by CCK-8 assay, and cell apoptosis was determined by flow cytometry. mRNA sequencing (mRNA-seq) was used to investigate the underlying mechanism of SENP3 knockdown on MCL development. A xenograft nude mouse model was established to evaluate the effect of SENP3 on MCL growth in vivo. RESULTS: SENP3 was upregulated in MCL patient samples and cells. Knockdown of SENP3 in MCL cells inhibited cell proliferation and promoted cell apoptosis. Meanwhile, the canonical Wnt signaling pathway and the expression of Wnt10a were suppressed after SENP3 knockdown. Furthermore, the growth of MCL cells in vivo was significantly inhibited after SENP3 knockdown in a xenograft nude mouse model. CONCLUSION: SENP3 participants in the development of MCL and may serve as a therapeutic target for MCL.

A role and mechanism for redox sensing by SENP1 in ß-cell responses to high fat feeding.

Pancreatic ß-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in ß-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted ß-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased ß-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or ß-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained ß-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

A naturally occurring G11S mutation in the 3C-like protease from the SARS-CoV-2 virus dramatically weakens the dimer interface.

The 3C-like protease (3CLpro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication.

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function.

Balanced cell division is secured by two different regulatory sites in OxyS RNA.

The hydrogen peroxide-induced small RNA OxyS has been proposed to originate from the 3' UTR of a peroxide mRNA. Unexpectedly, phylogenetic OxyS targetome predictions indicate that most OxyS targets belong to the category of "cell cycle," including cell division and cell elongation. Previously, we reported that Escherichia coli OxyS inhibits cell division by repressing expression of the essential transcription termination factor nusG, thereby leading to the expression of the KilR protein, which interferes with the function of the major cell division protein, FtsZ. By interfering with cell division, OxyS brings about cell-cycle arrest, thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division, OxyS inhibits mepS, which encodes an essential peptidoglycan endopeptidase that is responsible for cell elongation. Our study indicates that cell-cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress-induced sRNA OxyS.

SENP1 knockdown-mediated CTCF SUMOylation enhanced its stability and alleviated lipopolysaccharide-evoked inflammatory injury in human lung fibroblasts via regulation of FOXA2 transcription.

BACKGROUND: Excessive inflammation is the main cause of treatment failure in neonatal pneumonia (NP). CCCTC-binding factor (CTCF) represents an important node in various inflammatory diseases. In the present study, we tried to clarify the function and underlying molecular mechanism of CTCF on an in vitro cellular model of NP, which was generated by simulating the human lung fibroblast cell line WI-38 with lipopolysaccharide (LPS). METHODS: The SUMOylation level and protein interaction were verified by Co-immunoprecipitation assay. Cell viability was measured by Cell Counting Kit-8 assay. Inflammatory factors were examined by Enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by TUNEL assay. The binding activity of CTCF to target promoter was tested by chromatin immunoprecipitation and luciferase reporter assay. RESULTS: LPS treatment restrained cell viability, promoted the production of inflammatory factors, and enhanced cell apoptosis. CTCF overexpression played anti-inflammatory and anti-apoptotic roles. Furthermore, CTCF was modified by SUMOylation with small ubiquitin-like modifier protein 1 (SUMO1). Interfering with sumo-specific protease 1 (SENP1) facilitated CTCF SUMOylation and protein stability, thus suppressing LPS-evoked inflammatory and apoptotic injuries. Moreover, CTCF could bind to the forkhead box protein A2 (FOXA2) promoter region to promote FOXA2 expression. The anti-inflammatory and anti-apoptotic roles of CTCF are associated with FOXA2 activation. In addition, SENP1 knockdown increased FOXA2 expression by enhancing the abundance and binding ability of CTCF. CONCLUSIONS: SUMOylation of CTCF by SENP1 knockdown enhanced its protein stability and binding ability and it further alleviated LPS-evoked inflammatory injury in human lung fibroblasts by positively regulating FOXA2 transcription.

Deciphering the tumor-suppressive role of PSMB9 in melanoma through multi-omics and single-cell transcriptome analyses.

Skin cutaneous melanoma (SKCM) poses a significant challenge in skin cancers. Recent immunotherapy breakthroughs have revolutionized melanoma treamtment, yet tumor heterogeneity persists as an obstacle. Epigenetic modifications orchestrated by DNA methylation contributed to tumorigenesis, thus potentially unveiling melanoma prognosis. Here, we identified an interferon-gamma (IFN-g) sensitive subtype, which possesses favorable outcomes, robust infiltration CD8+T cells, and IFN-g score in bulk RNA-seq profile. Subsequently, we established an IFN-g sensitivity signature based on machine learning. We validated that PSMB9 is strongly correlated with immunotherapy response in both methylation and expression cohorts in this 10-probe signature. We assumed that PSMB9 acts as a putative melanoma suppressor, for its activation of CD8+T cell; capacity to modulate IFN-γ secretion; and dynamics altering IFN-g receptors in bulk tissue. We performed single-cell RNA-seq on immunotherapy patients' tissue to uncover the nuanced role of PSMB9 in activating CD8T + cells, enhancing IFN-g, and influencing malignant cells receptors and transcriptional factors. Overexpress PSMB9 in two SKCM cell lines to mimic the hypomethylated state to approve our conjecture. Strong cell proliferation and migration inhibition were detected on both cells, indicating that PSMB9 is present in tumor cells and that high expression is detrimental to tumor growth and migration. Overall, comprehensive integrated analysis shows that PSMB9 emerges as a vital prognostic marker, acting predictive potential regarding immunotherapy in melanoma. This evidence not only reveals the multifaceted impact of PSMB9 on both malignant and immune cells but also serves as a prospective target for undergoing immunotherapeutic strategies in the future.

[Interaction of SENP6 with PINK1 Promotes Temozolomide Resistance in Neuroglioma Cells via Inducing the Mitophagy].

Temozolomide resistance is a major cause of recurrence and poor prognosis in neuroglioma. Recently, growing evidence has suggested that mitophagy is involved in drug resistance in various tumor types. However, the role and molecular mechanisms of mitophagy in temozolomide resistance in glioma remain unclear. In this study, mitophagy levels in temozolomide-resistant and -sensitive cell lines were evaluated. The mechanisms underlying the regulation of mitophagy were explored through RNA sequencing, and the roles of differentially expressed genes in mitophagy and temozolomide resistance were investigated. We found that mitophagy promotes temozolomide resistance in glioma. Specifically, small ubiquitin-like modifier specific protease 6 (SENP6) promoted temozolomide resistance in glioma by inducing mitophagy. Protein-protein interactions between SENP6 and the mitophagy executive protein PTEN-induced kinase 1 (PINK1) resulted in a reduction in small ubiquitin-like modifier 2 (SUMO2)ylation of PINK1, thereby enhancing mitophagy. Our study demonstrates that by inducing mitophagy, the interaction of SENP6 with PINK1 promotes temozolomide resistance in glioblastoma. Therefore, targeting SENP6 or directly regulating mitophagy could be a potential and novel therapeutic target for reversing temozolomide resistance in glioma.

Myeloid SENP3 deficiency protects mice from diet and age-induced obesity via regulation of YAP1 SUMOylation.

Obesity is characterized by chronic low-grade inflammation, which is driven by macrophage infiltration in adipose tissue and leads to elevated cytokines such as interleukin-1ß (IL-1ß) in the circulation and tissues. Previous studies demonstrate that SENP3, a redox-sensitive SUMO2/3-specific protease, is strongly implicated in the development and progression of cancer and cardiovascular diseases. However, the role of SENP3 in obesity-associated inflammation remains largely unknown. To better understand the effects of SENP3 on adipose tissue macrophage (ATM) activation and function within the context of obesity, we generated mice with myeloid-specific deletion of SENP3 (Senp3flox/flox;Lyz2-Cre mice). We found that the expression of SENP3 is dramatically increased in ATMs during high-fat diet (HFD)-induced obesity in mice. Senp3flox/flox;Lyz2-Cre mice show lower body weight gain and reduced adiposity and adipocyte size after challenged with HFD and during aging. Myeloid-specific SENP3 deletion attenuates macrophage infiltration in adipose tissue and reduces serum levels of inflammatory factors during diet and age-induced obesity. Furthermore, we found that SENP3 knockout markedly inhibits cytokine release from macrophage after lipopolysaccharide and palmitic acid treatment in vitro. Mechanistically, in cultured peritoneal macrophages, SENP3 protein level is enhanced by IL-1ß, in parallel with the upregulation of Yes-associated protein 1 (YAP1). Moreover, we demonstrated that SENP3 modulates de-SUMO modification of YAP1 and SENP3 deletion abolishes the upregulation of YAP1 induced by IL-1ß. Most importantly, SENP3 deficiency reduces YAP1 protein level in adipose tissue during obesity. Our results highlight the important role of SENP3 in ATM inflammation and diet and age-induced obesity.

Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.

Spying on SARS-CoV-2 with Fluorescent Tags and Protease Reporters.

The SARS-CoV-2 coronavirus has caused worldwide disruption through the COVID-19 pandemic, providing a sobering reminder of the profound impact viruses can have on human well-being. Understanding virus life cycles and interactions with host cells lays the groundwork for exploring therapeutic strategies against virus-related diseases. Fluorescence microscopy plays a vital role in virus imaging, offering high spatiotemporal resolution, sensitivity, and spectroscopic versatility. In this opinion piece, we first highlight two recent techniques, SunTag and StayGold, for the in situ imaging of viral RNA translation and viral assembly. Next, we discuss a new class of genetically encoded fluorogenic protease reporters, such as FlipGFP, which can be customized to monitor SARS-CoV-2's main (Mpro) or papain-like (PLpro) protease activity. These assays have proven effective in identifying potential antivirals through high-throughput screening, making fluorogenic viral protease reporters a promising platform for viral disease diagnostics and therapeutics.

Asparagine endopeptidase protects podocytes in adriamycin-induced nephropathy by regulating actin dynamics through cleaving transgelin.

Focal segmental glomerulosclerosis (FSGS) is the most common glomerular disorder causing end-stage renal diseases worldwide. Central to the pathogenesis of FSGS is podocyte dysfunction, which is induced by diverse insults. However, the mechanism governing podocyte injury and repair remains largely unexplored. Asparagine endopeptidase (AEP), a lysosomal protease, regulates substrates by residue-specific cleavage or degradation. We identified the increased AEP expression in the primary proteinuria model which was induced by adriamycin (ADR) to mimic human FSGS. In vivo, global AEP knockout mice manifested increased injury-susceptibility of podocytes in ADR-induced nephropathy (ADRN). Podocyte-specific AEP knockout mice exhibited much more severe glomerular lesions and podocyte injury after ADR injection. In contrast, podocyte-specific augmentation of AEP in mice protected against ADRN. In vitro, knockdown and overexpression of AEP in human podocytes revealed the cytoprotection of AEP as a cytoskeleton regulator. Furthermore, transgelin, an actin-binding protein regulating actin dynamics, was cleaved by AEP, and, as a result, removed its actin-binding regulatory domain. The truncated transgelin regulated podocyte actin dynamics and repressed podocyte hypermotility, compared to the native full-length transgelin. Together, our data reveal a link between lysosomal protease AEP and podocyte cytoskeletal homeostasis, which suggests a potential therapeutic role for AEP in proteinuria disease.

Loss of ATG4B and ATG4A results in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy, which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but it is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors and a lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished processing of LC3B (also known as MAP1LC3B), and reduced GABARAP and GABARAPL1 levels, but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1-S phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.