Engineering strategies and challenges of endolysin as an antibacterial agent against Gram-negative bacteria.

Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique mechanism of hydrolysing peptidoglycans. Although endolysin exhibits excellent bactericidal effects on Gram-positive bacteria, the presence of the outer membrane of Gram-negative bacteria makes it difficult to lyse them extracellularly, thus limiting their application field. To enhance the extracellular activity of endolysin and facilitate its crossing through the outer membrane of Gram-negative bacteria, researchers have adopted physical, chemical, and molecular methods. This review summarizes the characterization of endolysin targeting Gram-negative bacteria, strategies for endolysin modification, and the challenges and future of engineering endolysin against Gram-negative bacteria in clinical applications, to promote the application of endolysin in the prevention and control of Gram-negative bacteria.

The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases.

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322-330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection.

68Ga-FAPI-04 PET/CT in Non-Small Cell Lung Cancer: Accurate Evaluation of Lymph Node Metastasis and Correlation with Fibroblast Activation Protein Expression.

Fibroblast activation protein (FAP) is a promising diagnostic and therapeutic target in various solid tumors. This study aimed to assess the diagnostic efficiency of 68Ga-labeled FAP inhibitor (FAPI)-04 PET/CT for detecting lymph node metastasis in non-small cell lung cancer (NSCLC) and to investigate the correlation between tumor 68Ga-FAPI-04 uptake and FAP expression. Methods: We retrospectively enrolled 136 participants with suspected or biopsy-confirmed NSCLC who underwent 68Ga-FAPI-04 PET/CT for initial staging. The diagnostic performance of 68Ga-FAPI-04 for the detection of NSCLC was evaluated. The final histopathology or typical imaging features were used as the reference standard. The SUVmax and SUVmean, 68Ga-FAPI-avid tumor volume (FTV), and total lesion FAP expression (TLF) were measured and calculated. FAP immunostaining of tissue specimens was performed. The correlation between 68Ga-FAPI-04 uptake and FAP expression was assessed using the Spearman correlation coefficient. Results: Ninety-one participants (median age, 65 y [interquartile range, 58-70 y]; 69 men) with NSCLC were finally analyzed. In lesion-based analysis, the diagnostic sensitivity and positive predictive value of 68Ga-FAPI-04 PET/CT for detection of the primary tumor were 96.70% (88/91) and 100% (88/88), respectively. In station-based analysis, the diagnostic sensitivity, specificity, and accuracy for the detection of lymph node metastasis were 72.00% (18/25), 93.10% (108/116), and 89.36% (126/141), respectively. Tumor 68Ga-FAPI-04 uptake (SUVmax, SUVmean, FTV, and TLF) correlated positively with FAP expression (r = 0.470, 0.477, 0.582, and 0.608, respectively; all P ≤ 0.001). The volume parameters FTV and TLF correlated strongly with FAP expression in 31 surgical specimens (r = 0.700 and 0.770, respectively; both P < 0.001). Conclusion: 68Ga-FAPI-04 PET/CT had excellent diagnostic efficiency for detecting lymph node metastasis, and 68Ga-FAPI-04 uptake showed a close association with FAP expression in participants with NSCLC.

Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Prediction of key amino acids of Salmonella phage endolysin LysST-3 and detection of its mutants' activity.

Salmonella Typhimurium, a zoonotic pathogen, causes systemic and localized infection. The emergence of drug-resistant S. Typhimurium has increased; treating bacterial infections remains challenging. Phage endolysins derived from phages have a broader spectrum of bacteriolysis and better bacteriolytic activity than phages, and are less likely to induce drug resistance than antibiotics. LysST-3, the endolysin of Salmonella phage ST-3, was chosen in our study for its high lytic activity, broad cleavage spectrum, excellent bioactivity, and moderate safety profile. LysST-3 is a promising antimicrobial agent for inhibiting the development of drug resistance in Salmonella. The aim of this study is to investigate the molecular characteristics of LysST-3 through the prediction of key amino acid sites of LysST-3 and detection of its mutants' activity. We investigated its lytic effect on Salmonella and identified its key amino acid sites of interaction with substrate. LysST-3 may be a Ca2+, Mg2+ - dependent metalloenzyme. Its concave structure of the bottom "gripper" was found to be an important part of its amino acid active site. We identified its key sites (29P, 30T, 86D, 88 L, and 89 V) for substrate binding and activity using amino acid-targeted mutagenesis. Alterations in these sites did not affect protein secondary structure, but led to a significant reduction in the cleavage activity of the mutant proteins. Our study provides a basis for phage endolysin modification to target drug-resistant bacteria. Identifying the key amino acid site of the endolysin LysST-3 provides theoretical support for the functional modification of the endolysin and the development of subsequent effective therapeutic solutions.

A dynamic ubiquitination balance of cell proliferation and endoreduplication regulators determines plant organ size.

Ubiquitination plays a crucial role throughout plant growth and development. The E3 ligase DA2 has been reported to activate the peptidase DA1 by ubiquitination, hereby limiting cell proliferation. However, the molecular mechanisms that regulate DA2 remain elusive. Here, we demonstrate that DA2 has a very high turnover and auto-ubiquitinates with K48-linkage polyubiquitin chains, which is counteracted by two deubiquitinating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13. Unexpectedly, we found that auto-ubiquitination of DA2 does not influence its stability but determines its E3 ligase activity. We also demonstrate that impairing the protease activity of DA1 abolishes the growth-reducing effect of DA2. Last, we show that synthetic, constitutively activated DA1-ubiquitin fusion proteins overrule this complex balance of ubiquitination and deubiquitination and strongly restrict growth and promote endoreduplication. Our findings highlight a nonproteolytic function of K48-linked polyubiquitination and reveal a mechanism by which DA2 auto-ubiquitination levels, in concert with UBP12 and UBP13, precisely monitor the activity of DA1 and fine-tune plant organ size.

SIAH1 modulates antiviral immune responses by targeting deubiquitinase USP19.

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.

Unravelling the Intricate Roles of FAM111A and FAM111B: From Protease-Mediated Cellular Processes to Disease Implications.

Proteases are critical enzymes in cellular processes which regulate intricate events like cellular proliferation, differentiation and apoptosis. This review highlights the multifaceted roles of the serine proteases FAM111A and FAM111B, exploring their impact on cellular functions and diseases. FAM111A is implicated in DNA replication and replication fork protection, thereby maintaining genome integrity. Additionally, FAM111A functions as an antiviral factor against DNA and RNA viruses. Apart from being involved in DNA repair, FAM111B, a paralog of FAM111A, participates in cell cycle regulation and apoptosis. It influences the apoptotic pathway by upregulating anti-apoptotic proteins and modulating cell cycle-related proteins. Furthermore, FAM111B's association with nucleoporins suggests its involvement in nucleo-cytoplasmic trafficking and plays a role in maintaining normal telomere length. FAM111A and FAM111B also exhibit some interconnectedness and functional similarity despite their distinct roles in cellular processes and associated diseases resulting from their dysfunction. FAM111A and FAM111B dysregulation are linked to genetic disorders: Kenny-Caffey Syndrome type 2 and Gracile Bone Dysplasia for FAM111A and POIKTMP, respectively, and cancers. Therefore, the dysregulation of these proteases in diseases emphasizes their potential as diagnostic markers and therapeutic targets. Future research is essential to unravel the intricate mechanisms governing FAM111A and FAM111B and explore their therapeutic implications comprehensively.

Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

Revealing extracellular protein profile and excavating spoilage-related proteases of Aeromonas salmonicida based on multi-omics investigation.

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida, and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB, hap, pepA, ftsI, and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development.

Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli.

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the 'space-making activity' is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.

Gram-negative endolysins: overcoming the outer membrane obstacle.

Our ability to control the growth of Gram-negative bacterial pathogens is challenged by rising antimicrobial resistance and requires new approaches. Endolysins are phage-derived enzymes that degrade peptidoglycan and therefore offer potential as antimicrobial agents. However, the outer membrane (OM) of Gram-negative bacteria impedes the access of externally applied endolysins to peptidoglycan. This review highlights recent advances in the discovery and characterization of natural endolysins that can breach the OM, as well as chemical and engineering approaches that increase antimicrobial efficacy of endolysins against Gram-negative pathogens.

Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1ß to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1ß (Nsp1ß) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1ß protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1ß (Nsp1ß) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1ß, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1ß, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Chromatin-binding deubiquitinase MYSM1 acts in haematopoietic progenitors to control dendritic cell development and to program dendritic cell responses to microbial stimulation.

Dendritic cells (DCs) are the most significant antigen presenting cells of the immune system, critical for the activation of naïve T cells. The pathways controlling DC development, maturation, and effector function therefore require precise regulation to allow for an effective induction of adaptive immune response. MYSM1 is a chromatin binding deubiquitinase (DUB) and an activator of gene expression via its catalytic activity for monoubiquitinated histone H2A (H2A-K119ub), which is a highly abundant repressive epigenetic mark. MYSM1 is an important regulator of haematopoiesis in mouse and human, and a systemic constitutive loss of Mysm1 in mice results in a depletion of many haematopoietic progenitors, including DC precursors, with the downstream loss of most DC lineage cells. However, the roles of MYSM1 at the later checkpoints in DC development, maturation, activation, and effector function at present remain unknown. In the current work, using a range of novel mouse models (Mysm1flCreERT2, Mysm1flCD11c-cre, Mysm1DN), we further the understanding of MYSM1 functions in the DC lineage: assessing the requirement for MYSM1 in DC development independently of other complex developmental phenotypes, exploring its role at the later checkpoints in DC maintenance and activation in response to microbial stimulation, and testing the requirement for the DUB catalytic activity of MYSM1 in these processes. Surprisingly, we demonstrate that MYSM1 expression and catalytic activity in DCs are dispensable for the maintenance of DC numbers in vivo or for DC activation in response to microbial stimulation. In contrast, MYSM1 acts via its DUB catalytic activity specifically in haematopoietic progenitors to allow normal DC lineage development, and its loss results not only in a severe DC depletion but also in the production of functionally altered DCs, with a dysregulation of many housekeeping transcriptional programs and significantly altered responses to microbial stimulation.

SARS-CoV-2 and oncolytic EV-D68-encoded proteases differentially regulate pyroptosis.

Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.

Ubiquitin-specific Protease 35 Promotes Gastric Cancer Metastasis by Increasing the Stability of Snail1.

Deubiquitinase (DUB) dysregulation is closely associated with multiple diseases, including tumors. In this study, we used data from The Cancer Genome Atlas and Gene Expression Omnibus databases to analyze the expression of 51 ubiquitin-specific proteases (USPs) in gastric cancer (GC) tissues and adjacent non-neoplastic tissues. The Kaplan-Meier Plotter database was used to analyze the association of the differentially expressed USPs with the overall survival of patients with GC. The results showed that five USPs (USP5, USP10, USP13, USP21, and USP35) were highly expressed in GC tissues and were associated with poor prognosis in patients with GC. Because the epithelial-mesenchymal transition enables epithelial cells to acquire mesenchymal features and contributes to poor prognosis, we investigated whether these USPs had regulatory effects on the key epithelial-mesenchymal transition transcription factor Snail1. Our results showed that USP35 exhibited the most significant regulation on Snail1. Overexpression of USP35 increased and its knockdown decreased Snail1 protein levels. Mechanistically, USP35 interacted with Snail1 and removed its polyubiquitinated chain, thereby increasing its stability. Furthermore, USP35 promoted the invasion and migration of GC cells depending on its DUB activity. USP35 knockdown exhibited the opposite effect. Snail1 depletion partially abrogated the biological effects of USP35. Experiments using nude mouse tail vein injections indicated that wild-type USP35, but not the catalytically inactive USP35-C450A mutant, dramatically enhanced cell colonization and tumorigenesis in the lungs of mice. In addition, USP35 positively correlated with Snail1 expression in clinical GC tissues. Helicobacter pylori infection increased USP35 and Snail1 expression levels. Altogether, we found that USP35 can deubiquitinate Snail1 and increase its expression, thereby contributing to the malignant progression of GC. Therefore, USP35 may serve as a viable target for GC treatment.

Endolysin EN572-5 as an alternative to treat urinary tract infection caused by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an opportunistic pathogen causing urinary tract infection (UTI). Endolysin EN572-5 was identified in prophage KMB-572-E of the human isolate Streptococcus agalactiae KMB-572. The entire EN572-5 gene was cloned into an expression vector and the corresponding recombinant protein EN572-5 was expressed in Escherichia coli in a soluble form, isolated by affinity chromatography, and characterized. The isolated protein was highly active after 30 min incubation in a temperature range of - 20 °C to 37 °C and in a pH range of 5.5-8.0. The endolysin EN572-5 lytic activity was tested on different Streptococcus spp. and Lactobacillus spp. The enzyme lysed clinical GBS (n = 31/31) and different streptococci (n = 6/8), and also exhibited moderate lytic activity against UPEC (n = 4/4), but no lysis of beneficial vaginal lactobacilli (n = 4) was observed. The ability of EN572-5 to eliminate GBS during UTI was investigated using an in vitro model of UPSA. After the administration of 3 µM EN572-5, a nearly 3-log decrease of urine bacterial burden was detected within 3 h. To date, no studies have been published on the use of endolysins against S. agalactiae during UTI. KEY POINTS: • A lytic protein, EN572-5, from a prophage of a human GBS isolate has been identified. • This protein is easily produced, simple to prepare, and stable after lyophilization. • The bacteriolytic activity of EN572-5 was demonstrated for the first time in human urine.

A GH19 lysozyme and peptidase from Myoviridae cyanophages lacking the typical holin-endolysin system exhibit lytic activity.

Most of the dsDNA cyanophages employ holin-endolysin lysis systems to damage the host cells. This study aimed to elucidate the lytic activity of ORF91 and ORF117 in the cyanophage MaMV-DH01, which lacked a conventional cholinesterase system. These two proteins contained Lyz-like superfamily domains and were annotated as a member of GH family 19 (named DHGH19) and peptidase (named DHpeptidase), respectively. Overexpression of DHGH19 in E. coli over a 5 h course demonstrated potent bactericidal activity, evident from significant growth inhibition, membrane damage, and leakage of intracellular enzymes of E. coli cells. However, the lytic activity of DHpeptidase was relatively weaker, exhibiting a bacteriostatic effect. It was important to highlight that the specific mutation of enzyme-catalyzed residues in DHGH19 (E122 and E131) showed that these were the essential amino acids for DHGH19 to exert its bactericidal activity. Furthermore, the lytic function of DHGH19 and DHpeptidase on cyanobacteria cells was confirmed by their overexpression in the cyanobacterium Synechocystis sp. PCC6803. Overall, this study provides novel insights into the lytic mechanism of Myoviridae cyanophage, offering potential alternatives for the development of GH19 and peptidase as new antibacterial agents in the future.

USP5: Comprehensive insights into structure, function, biological and disease-related implications, and emerging therapeutic opportunities.

Ubiquitin specific protease 5 (USP5) is a vital deubiquitinating enzyme that regulates various physiological functions by removing ubiquitin chains from target proteins. This review provides an overview of the structural and functional characteristics of USP5. Additionally, we discuss the role of USP5 in regulating diverse cellular processes, including cell proliferation, apoptosis, DNA double-strand damage, methylation, heat stress, and protein quality control, by targeting different substrates. Furthermore, we describe the involvement of USP5 in several pathological conditions such as tumors, pathological pain, developmental abnormalities, inflammatory diseases, and virus infection. Finally, we introduce newly developed inhibitors of USP5. In conclusion, investigating the novel functions and substrates of USP5, elucidating the underlying mechanisms of USP5-substrate interactions, intensifying the development of inhibitors, and exploring the upstream regulatory mechanisms of USP5 in detail can provide a new theoretical basis for the treatment of various diseases, including cancer, which is a promising research direction with considerable potential. Overall, USP5 plays a critical role in regulating various physiological and pathological processes, and investigating its novel functions and regulatory mechanisms may have significant implications for the development of therapeutic strategies for cancer and other diseases.