Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus.

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

Fibroblast Activation Protein Is Expressed by Altered Osteoprogenitors and Associated to Disease Burden in Fibrous Dysplasia.

Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.

Differential processing of VesB by two rhomboid proteases in Vibrio cholerae.

Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM. IMPORTANCE: Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae. This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria.

A Clostridioides difficile endolysin modulates toxin secretion without cell lysis.

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404.

TWIST1+FAP+ fibroblasts in the pathogenesis of intestinal fibrosis in Crohn's disease.

Intestinal fibrosis, a severe complication of Crohn's disease (CD), is characterized by excessive extracellular matrix (ECM) deposition and induces intestinal strictures, but there are no effective antifibrosis drugs available for clinical application. We performed single-cell RNA sequencing (scRNA-Seq) of fibrotic and nonfibrotic ileal tissues from patients with CD with intestinal obstruction. Analysis revealed mesenchymal stromal cells (MSCs) as the major producers of ECM and the increased infiltration of its subset FAP+ fibroblasts in fibrotic sites, which was confirmed by immunofluorescence and flow cytometry. Single-cell transcriptomic profiling of chronic dextran sulfate sodium salt murine colitis model revealed that CD81+Pi16- fibroblasts exhibited transcriptomic and functional similarities to human FAP+ fibroblasts. Consistently, FAP+ fibroblasts were identified as the key subtype with the highest level of ECM production in fibrotic intestines. Furthermore, specific knockout or pharmacological inhibition of TWIST1, which was highly expressed by FAP+ fibroblasts, could significantly ameliorate fibrosis in mice. In addition, TWIST1 expression was induced by CXCL9+ macrophages enriched in fibrotic tissues via IL-1ß and TGF-ß signal. These findings suggest the inhibition of TWIST1 as a promising strategy for CD fibrosis treatment.

O-Sialoglycoprotein Endopeptidase Deficiency Impairs Proteostasis and Induces Autophagy in Human Embryonic Stem Cells.

The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway-Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.

Fibroblast activation protein (FAP) as a prognostic biomarker in multiple tumors and its therapeutic potential in head and neck squamous cell carcinoma.

Background: Fibroblast activation protein (FAP), a cell surface serine protease, plays roles in tumor invasion and immune regulation. However, there is currently no pan-cancer analysis of FAP. Objective: We aimed to assess the pan-cancer expression profile of FAP, its molecular function, and its potential role in head and neck squamous cell carcinoma (HNSC). Methods: We analyzed gene expression, survival status, immune infiltration, and molecular functional pathways of FAP in The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) tumors. Furthermore, to elucidate the role of FAP in HNSC, we performed proliferation, migration, and invasion assays post-FAP overexpression or knock-down. Results: FAP expression was elevated in nine tumor types and was associated with poor survival in eight of them. In the context of immune infiltration, FAP expression negatively correlated with CD8+ T-cell infiltration in five tumor types and positively with regulatory T-cell infiltration in four tumor types. Our enrichment analysis highlighted FAP's involvement in the PI3K-Akt signaling pathway. In HNSC cells, FAP overexpression activated the PI3K-Akt pathway, promoting tumor proliferation, migration, and invasion. Conversely, FAP knockdown showed inhibitory effects. Conclusion: Our study unveils the association of FAP with poor tumor prognosis across multiple cancers and highlights its potential as a therapeutic target in HNSC.

Determinants in the phage life cycle: The dynamic nature of ssDNA phage FLiP and host interactions under varying environmental conditions and growth phases.

The influence of environmental factors on the interactions between phages and bacteria, particularly single-stranded DNA (ssDNA) phages, has been largely unexplored. In this study, we used Finnlakevirus FLiP, the first known ssDNA phage species with a lipid membrane, as our model phage. We examined the infectivity of FLiP with three Flavobacterium host strains, B330, B167 and B114. We discovered that FLiP infection is contingent on the host strain and conditions such as temperature and bacterial growth phase. FLiP can infect its hosts across a wide temperature range, but optimal phage replication varies with each host. We uncovered some unique aspects of phage infectivity: FLiP has limited infectivity in liquid-suspended cells, but it improves when cells are surface-attached. Moreover, FLiP infects stationary phase B167 and B114 cells more rapidly and efficiently than exponentially growing cells, a pattern not observed with the B330 host. We also present the first experimental evidence of endolysin function in ssDNA phages. The activity of FLiP's lytic enzymes was found to be condition-dependent. Our findings underscore the importance of studying phage ecology in contexts that are relevant to the environment, as both the host and the surrounding conditions can significantly alter the outcome of phage-host interactions.

Antibacterial activity of bacteriophage-encoded endolysins against planktonic and biofilm cells of pathogenic Escherichia coli.

This study was designed to assess the possibility of using bacteriophage-encoded endolysins for controlling planktonic and biofilm cells. The endolysins, LysEP114 and LysEP135, were obtained from plasmid vectors containing the endolysin genes derived from Escherichia coli phages. The high identity (>96 %) was observed between LysEP114 and LysEP135. LysEP114 and LysEP135 were characterized by pH, thermal, and lactic acid stability, lytic spectrum, antibacterial activity, and biofilm eradication. The molecular masses of LysEP114 and LysEP135 were 18.2 kDa, identified as muramidases. LysEP114 and LysEP135 showed high lytic activity against the outer membrane-permeabilized E. coli KCCM 40405 at below 37 °C, between pH 5 to 11, and below 70 mM of lactic acid. LysEP114 and LysEP135 showed the broad rang of lytic activity against E. coli KACC 10115, S. Typhimurium KCCM 40253, S. Typhimurium CCARM 8009, tetracycline-resistant S. Typhimurium, polymyxin B-resistant S. Typhimurium, chloramphenicol-resistant S. Typhimurium, K. pneumoniae ATCC 23357, K. pneumoniae CCARM 10237, and Shigella boydii KACC 10792. LysEP114 and LysEP135 effectively reduced the numbers of planktonic E. coli KCCM by 1.7 and 2.1 log, respectively, when treated with 50 mM lactic acid. The numbers of biofilm cells were reduced from 7.3 to 4.1 log CFU/ml and 2.2 log CFU/ml, respectively, when treated with LysEP114- and LysEP135 in the presence of 50 mM lactic acid. The results suggest that the endolysins in combination with lactic acid could be potential alternative therapeutic agents for controlling planktonic and biofilm cells.

Construing the function of N-terminal domain of D29 mycobacteriophage LysA endolysin in phage lytic efficiency and proliferation.

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N-terminal domain [NTD] and lysozyme-like domain [LD]) and a C-terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD-deficient D29 (D29∆NTD) by deleting the NTD-coding region from D29 genome using CRISPY-BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD-mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD-deficient LysA produced by D29∆NTD, despite having catalytically-active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells.

OBJECTIVE: Obesity-induced kidney injury contributes to the development of diabetic nephropathy (DN). Here, we identified the functions of ubiquitin-specific peptidase 19 (USP19) in HK-2 cells exposed to a combination of high glucose (HG) and free fatty acid (FFA) and determined its association with TGF-beta-activated kinase 1 (TAK1). METHODS: HK-2 cells were exposed to a combination of HG and FFA. USP19 mRNA expression was detected by quantitative RT-PCR (qRT-PCR), and protein analysis was performed by immunoblotting (IB). Cell growth was assessed by Cell Counting Kit-8 (CCK-8) viability and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Cell cycle distribution and apoptosis were detected by flow cytometry. The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation (Co-IP) assays and IB. RESULTS: In HG+FFA-challenged HK-2 cells, USP19 was highly expressed. USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells. Moreover, USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1 (PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species (ROS) generation in HK-2 cells. Mechanistically, USP19 stabilized the TAK1 protein through deubiquitination. Importantly, increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells. CONCLUSION: The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1, providing a potential therapeutic strategy for combating DN.

Intercellular interaction between FAP+ fibroblasts and CD150+ inflammatory monocytes mediates fibrostenosis in Crohn's disease.

Crohn's disease (CD) is marked by recurring intestinal inflammation and tissue injury, often resulting in fibrostenosis and bowel obstruction, necessitating surgical intervention with high recurrence rates. To elucidate the mechanisms underlying fibrostenosis in CD, we analyzed the transcriptome of cells isolated from the transmural ileum of patients with CD, including a trio of lesions from each patient: non-affected, inflamed, and stenotic ileum samples, and compared them with samples from patients without CD. Our computational analysis revealed that profibrotic signals from a subset of monocyte-derived cells expressing CD150 induced a disease-specific fibroblast population, resulting in chronic inflammation and tissue fibrosis. The transcription factor TWIST1 was identified as a key modulator of fibroblast activation and extracellular matrix (ECM) deposition. Genetic and pharmacological inhibition of TWIST1 prevents fibroblast activation, reducing ECM production and collagen deposition. Our findings suggest that the myeloid-stromal axis may offer a promising therapeutic target to prevent fibrostenosis in CD.

The effectiveness of endolysin ENDO-1252 from Salmonella bacteriophage-1252 against nontyphoidal Salmonella enterica.

Salmonella enterica (S. enterica) is the most common food and waterborne pathogen worldwide. The growing trend of antibiotic-resistant S. enterica poses severe healthcare threats. As an alternative antimicrobial agent, bacteriophage-encoded endolysins (endolysins) are a potential agent in controlling S. enterica infection. Endolysins are enzymes that particularly target the peptidoglycan layer of bacterial cells, leading to their rupture and destruction. However, the application of endolysins against Gram-negative bacteria is limited due to the presence of the outer membrane in the cell wall, which hinders the permeation of externally applied endolysins. This study aimed the prokaryotic expression system to produce the recombinant endolysin ENDO-1252, encoded by the Salmonella bacteriophage-1252 associated with S. Enteritidis. Subsequently, ENDO-1252 had strong lytic activity not only against S. Enteritidis but also against S. Typhimurium. In addition, ENDO-1252 showed optimal thermostability and lytic activity at 25°C with a pH of 7.0. In combination with 0.1 mM EDTA, the effect of 120 µg of ENDO-1252 for 6 hours exhibited the highest lytic activity, resulting in a reduction of 1.15 log or 92.87% on S. Enteritidis. These findings suggest that ENDO-1252 can be used as a potential and innovative antibacterial agent for controlling the growth of S. Enteritidis.

Structural analysis of the peptidoglycan DL-endopeptidase CwlO complexed with its inhibitory protein IseA.

Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA.

FAP positive cancer-associated fibroblasts promote tumor progression and radioresistance in esophageal squamous cell carcinoma by transferring exosomal lncRNA AFAP1-AS1.

Cancer-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in the tumor microenvironment, which play important roles in regulating tumor progression and therapy resistance by transferring exosomes to cancer cells. However, how CAFs modulate esophageal squamous cell carcinoma (ESCC) progression and radioresistance remains incompletely understood. The expression of fibroblast activation protein (FAP) in CAFs was evaluated by immunohistochemistry in 174 ESCC patients who underwent surgery and 78 pretreatment biopsy specimens of ESCC patients who underwent definitive chemoradiotherapy. We sorted CAFs according to FAP expression, and the conditioned medium (CM) was collected to culture ESCC cells. The expression levels of several lncRNAs that were considered to regulate ESCC progression and/or radioresistance were measured in exosomes derived from FAP+ CAFs and FAP- CAFs. Subsequently, cell counting kit-8, 5-ethynyl-2'-deoxyuridine, transwell, colony formation, and xenograft assays were performed to investigate the functional differences between FAP+ CAFs and FAP- CAFs. Finally, a series of in vitro and in vivo assays were used to evaluate the effect of AFAP1-AS1 on radiosensitivity of ESCC cells. FAP expression in stromal CAFs was positively correlated with nerve invasion, vascular invasion, depth of invasion, lymph node metastasis, lack of clinical complete response and poor survival. Culture of ESCC cells with CM/FAP+ CAFs significantly increased cancer proliferation, migration, invasion and radioresistance, compared with culture with CM/FAP- CAFs. Importantly, FAP+ CAFs exert their roles by directly transferring the functional lncRNA AFAP1-AS1 to ESCC cells via exosomes. Functional studies showed that AFAP1-AS1 promoted radioresistance by enhancing DNA damage repair in ESCC cells. Clinically, high levels of plasma AFAP1-AS1 correlated with poor responses to dCRT in ESCC patients. Our findings demonstrated that FAP+ CAFs promoted radioresistance in ESCC cells through transferring exosomal lncRNA AFAP1-AS1; and may be a potential therapeutic target for ESCC treatment.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.

BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient "bioPROTAC" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.

Interleukin-17A educated hepatic stellate cells promote hepatocellular carcinoma occurrence through fibroblast activation protein expression.

BACKGROUND: Liver cancer is typified by a complex inflammatory tumor microenvironment, where an array of cytokines and stromal cells orchestrate a milieu that significantly influences tumorigenesis. Interleukin-17A (IL-17A), a pivotal pro-inflammatory cytokine predominantly secreted by Th17 cells, is known to play a substantial role in the etiology and progression of liver cancer. However, the precise mechanism by which IL-17A engages with hepatic stellate cells (HSCs) to facilitate the development of hepatocellular carcinoma (HCC) remains to be fully elucidated. This investigation seeks to unravel the interplay between IL-17A and HSCs in the context of HCC. METHODS: An HCC model was established in male Sprague-Dawley rats using diethylnitrosamine to explore the roles of IL-17A and HSCs in HCC pathogenesis. In vivo overexpression of Il17a was achieved using adeno-associated virus. A suite of molecular techniques, including RT-qPCR, enzyme-linked immunosorbent assays, Western blotting, cell counting kit-8 assays and colony formation assays, was employed for in vitro analyses. RESULTS: The study findings indicate that IL-17A is a key mediator in HCC promotion, primarily through the activation of hepatic progenitor cells (HPCs). This pro-tumorigenic influence appears to be mediated by HSCs, rather than through a direct effect on HPCs. Notably, IL-17A-induced expression of fibroblast activation protein (FAP) in HSCs emerged as a critical factor in HCC progression. Silencing Fap in IL-17A-stimulated HSCs was observed to reverse the HCC-promoting effects of HSCs. CONCLUSIONS: The collective evidence from this study implicates the IL-17A/FAP signaling axis within HSCs as a contributor to HCC development by enhancing HPC activation. These findings bolster the potential of IL-17A as a diagnostic and preventative target for HCC, offering new avenues for therapeutic intervention.

From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).