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Purpose: To evaluate the effect of urethane methacrylate precursor (UMP) on the enzymatic resistance of demineralized dentin (DD) matrices. Materials and Methods: Experimental treatments containing 0 (control), 1, and 5 mmol/L UMP dissolved in an acetone (Ace) solution were formulated. Dentin matrix specimens were demineralized in vitro and immersed in the experimental treatments for 1 h. The treated specimens were then stored in 0.1 mg/mL collagenase solution for 24 h, after which their dry mass loss and hydroxyproline (HYP) release were assessed. The swelling ratios of specimens in each group were also evaluated. The interaction between UMP and the dentin matrix was observed using field-emission scanning electron microscopy (FE-SEM). Endogenous enzyme activity in dentin was evaluated using confocal laser scanning microscopy (CLSM). Results: Compared with the other treatment groups, treatment with 1 mM and 5 mM UMP-Ace significantly decreased the dry mass loss, HYP release and swelling ratio of the DD matrix (p < 0.05). FE-SEM and CLSM observations showed that treatment with UMP-Ace protected the structure of the dentin matrix and decreased porosity within the dentin-collagen network. Conclusion: Treatment with 1 mM and 5 mM UMP-Ace protects DD matrix against collagenase degradation and may be clinically useful for improving the durability of the hybrid layer. [ABSTRACT FROM AUTHOR]

Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments. Synopsis: Functional protein studies within their native regulatory context and at physiological expression levels require endogenous tagging via gene editing approaches. This study establishes a streamlined system, qTAG, which offers an adaptable set of optimized CRISPR repair cassettes enabling diverse applications. The qTAG system offers cassette designs for precise N- or C-terminal tagging. qTAG cassettes support targeting of both the HDR and MMEJ pathways, with flanking cloning sites for integrating long or short homology arms. Strategic restriction sites within cassettes allow easy customization of selectable markers and functional tags. Incorporated lox sites enable post-integration removal of marker genes. The adaptable cassette structure permits payload re-designing for alternative applications, expanding the toolkit's versatility. An new set of optimized repair cassettes for precise N- or C-terminal tagging and facile marker gene removal enables diverse applications in functional protein studies in cells. [ABSTRACT FROM AUTHOR]

Pollen development and germination are critical for successful generation of offspring in plants, yet they are highly susceptible to heat stress (HS). However, the molecular mechanism underlying this process has not been fully elucidated. In this study, we highlight the essential roles of two mRNA capping enzymes, named Arabidopsis mRNA capping phosphatase (ARCP) 1 and 2, in regulating male and female gamete development. The transmission efficiencies of gametes carrying arcp1 arcp2 from arcp1+/−arcp2−/− and arcp1−/−arcp2+/− mutants are 30% and zero, respectively. These mutants exhibited a significant increase in misshaped pollen, with germination rates approximately half of those in wild type. ARCP1/2 exhibit RNA triphosphatase and RNA guanylyltransferase activities, which are required for proper pollen development. Through RNA‐seq analysis, genes involved in pollen development/germination and HS response were identified as downregulated genes in pollen from arcp1+/−arcp2−/− mutant. Furthermore, ARCP2 protein is degraded under HS condition, and inducing the expression of ARCP2 can increase the pollen germination rate under elevated temperature. We propose that HS triggers the degradation of mRNA capping enzymes, which in turn disrupts the transcriptome that required for pollen development and pollen germination and ultimately leads to male sterility. Summary statement: Two mRNA capping enzymes, ARCP1 and ARCP2, are essential for gametophyte development and pollen germination in Arabidopsis.Heat shock induces the degradation of ARCP2 protein, which affects transcript level of critical genes function in pollen development/germination thus causing male sterility. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a life-threatening syndrome associated with hyperproliferation of pulmonary artery smooth muscle cells (PASMCs), which exhibit features similar to those of cancer cells. Currently, there is no curative treatment for PH. LKB1 is known as a tumor suppressor gene with an antiproliferative effect on cancer cells. However, its role and mechanism in the development of PH remain unclear. Gain- and loss-of-function strategies were used to elucidate the mechanisms of LKB1 in regulating the occurrence and progression of PH. Sugen 5416/hypoxia (SuHx) PH model was utilized for in vivo study. We observed a decreased expression of LKB1 not only in the lung vessels of the SuHx mouse model but also in human PASMCs (HPASMCs) exposed to hypoxia. Smooth muscle–specific LKB1 knockout significantly aggravated SuHx-induced PH in mice. RNA-sequencing analysis revealed a substantial increase in bone morphogenetic protein 4 (BMP4) in the aortas of LKB1SMKO mice compared with controls, identifying BMP4 as a novel target of LKB1. LKB1 knockdown in HPASMCs cultured under hypoxic conditions increased BMP4 protein level and HPASMC proliferation and migration. The coimmunoprecipitation analysis revealed that LKB1 directly modulates BMP4 protein degradation through phosphorylation. Therapeutically, suppressing BMP4 expression in smooth muscle cells alleviates PH in LKB1SMKO mice. Our findings demonstrate that LKB1 attenuates PH by enhancing the lysosomal degradation of BMP4, thus suppressing the proliferation and migration of HPASMCs. Modulating the LKB1-BMP4 axis in smooth muscle cells could be a promising therapeutic strategy of PH. [ABSTRACT FROM AUTHOR]

Background: Continuous fermentation offers advantages in improving production efficiency and reducing costs, making it highly competitive for industrial ethanol production. A key requirement for Saccharomyces cerevisiae strains used in this process is their tolerance to high ethanol concentrations, which enables them to adapt to continuous fermentation conditions. To explore how yeast cells respond to varying levels of ethanol stress during fermentation, a two-month continuous fermentation was conducted. Cells were collected at different ethanol concentrations (from 60 g/L to 100 g/L) for comparative transcriptomic analysis. Results: During continuous fermentation, as ethanol concentration increased, the expression of genes associated with cytoplasmic ribosomes, translation, and fatty acid biosynthesis progressively declined, while the expression of genes related to heat shock proteins (HSPs) and ubiquitin-mediated protein degradation gradually increased. Besides, cells exhibited distinct responses to varying ethanol concentrations. At lower ethanol concentrations (nearly 70 g/L), genes involved in mitochondrial ribosomes, oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, antioxidant enzymes, ergosterol synthesis, and glycerol biosynthesis were specifically upregulated compared to those at 60 g/L. This suggests that cells enhanced respiratory energy production, ROS scavenging capacity, and the synthesis of ergosterol and glycerol to counteract stress. At relatively higher ethanol concentrations (nearly 80 g/L), genes involved in respiration and ergosterol synthesis were inhibited, while those associated with glycolysis and glycerol biosynthesis were notably upregulated. This suggests a metabolic shift from respiration towards enhanced glycerol synthesis. Interestingly, the longevity-regulating pathway seemed to play a pivotal role in mediating the cellular adaptations to different ethanol concentrations. Upon reaching an ethanol concentration of 100 g/L, the aforementioned metabolic activities were largely inhibited. Cells primarily focused on enhancing the clearance of denatured proteins to preserve cellular viability. Conclusions: This study elucidated the mechanisms by which an ethanol-tolerant S. cerevisiae strain adapts to increasing ethanol concentrations during continuous fermentation. The findings suggest that the longevity-regulating pathway may play a critical role in adapting to varying ethanol stress by regulating mitochondrial respiration, glycerol synthesis, ergosterol synthesis, antioxidant enzyme, and HSPs. This work provides a novel and valuable understanding of the mechanisms that govern ethanol tolerance during continuous fermentation. [ABSTRACT FROM AUTHOR]

Aberrant autophagy in vascular smooth muscle cells (VSMCs) is associated with the progression of vascular remodeling diseases caused by neointimal hyperplasia. Platelet-derived growth factor-BB (PDGF-BB)-induced vascular remodeling is accompanied by autophagy activation, however, the involvement of circular RNAs (circRNAs) remains unclear. Here, we show the role of PDGF-BB-regulated hsa_circ_0001304 (circ-1304) in neointimal hyperplasia and its potential involvement in VSMC autophagy, while also elucidating the potential mechanisms. Functionally, overexpression of circ-1304 promotes VSMC autophagy in vitro and exacerbates neointimal hyperplasia in vivo, and this exacerbation is accompanied by autophagy activation. Mechanistically, circ-1304 acts as a sponge for miR-636, resulting in increased protein levels of YTHDF2. Subsequently, the YTHDF2 protein promotes the degradation of mTOR mRNA by binding to the latter's m6A modification sites. We demonstrate that PDGF-BB activates VSMC autophagy via circRNA regulation. Therefore, circ-1304 may serve as a potential therapeutic target for vascular remodeling diseases. Hsa_circ_0001304 relies on miR-636 to target the YTHDF2/mTOR axis, thereby activating VSMC autophagy and exacerbating vascular neointimal hyperplasia. [ABSTRACT FROM AUTHOR]

The VHL-containing cullin-RING E3 ubiquitin ligase (CRL2VHL) complex is an E3 ligase commonly used in targeted protein degradation (TPD). Hydroxyproline-based ligands that mimic VHL substrates have been developed as anchor molecules for proteolysis-targeting chimeras (PROTACs) in TPD. To expand the chemical space for VHL ligands, we conducted fragment screening using VHL–ELOB–ELOC (VBC) proteins. We found that certain 7-hydroxycoumarin derivatives (7HCs), rather than VHL, would bind to the ELOC component of the VBC complex. The 7HC binding site overlapped with the CUL2 binding interface on ELOC but did not overlap with the CUL5 binding interface, suggesting that 7HCs may influence the formation of CRL2 but not CRL5. Although the binding affinities of these 7HCs to the VBC complex were relatively low, they represent novel and promising foundational agents for the development of chemical probes or inhibitors that target ELOC-containing CRLs. [ABSTRACT FROM AUTHOR]

Introduction: A small number of soybean allergens [including Glycinin (11S) and β-Conglycinin (7S)] in the commercially available corn-soybean meal diet can still cause allergy in some weaned piglets, which may be the result of the interaction of genetic, and nutrition, but the specific mechanism is still unclear. Methods: In this study, 20 allergic piglets and 20 non-allergic piglets were selected from 92 weaned piglets by skin sensitization tests, which were used to examine the whole sequence genome. The indicators related to humoral and cellular immunity, transcriptomics, and metabolomics analysis were determined by randomly selecting 5 boars in the allergic group and non-allergic group and then performing a validation in vitro. Results: The sensitization rate of soybean antigen in the corn-soybean meal diet was 21.74% and there was a gender difference with the sensitization rate of female pigs (31.34%) being higher than that of male pigs (13.23%). Moreover, the levels of inflammatory factors (IL-1β, IL-4, TNF-α) and antibodies (IgG, IgE, and specific IgG) in allergic piglets were significantly higher than those in non-allergic piglets (P < 0.05). Whole genome re-sequencing analysis revealed specific mutations in the exons and URT5 of TRAPPC2, PIR, CFP, and SOWAHD genes and showed significantly higher expression levels of related genes in the spleen of allergic piglets (P < 0.05). Transcriptome analysis identified IL17REL, CCL19, CD1E, CD1.1, etc. immune differential genes, metabolomics results showed that soybean antigen affected the utilization and metabolism of intestinal nutrients in piglets, mainly the digestion and absorption of protein and the synthesis and metabolism of amino acids. Transfection of CFP/TRAPPC2/CCL19 siRNA could partially alleviate the injury of RAW264.7 cells or IPEC-J2 cells induced by β-Conglycinin. Conclusion: Therefore, the individual differences in intestinal damage induced by soybean antigen protein in the corn-soybean meal diet are closely related to PIR, CFP, TRAPPC2, SOWAHD, and CCL19 genes. Soybean antigens affect the intestinal nutrient utilization and metabolism of piglets, which provides a scientific reference for the study of soybean antigen sensitization mechanisms, precision nutrition, disease prevention, and control of piglets, and also lays a foundation for human foodborne diseases. [ABSTRACT FROM AUTHOR]

Aboveground plant material is subject to stress-induced senescence after harvest. For flowers, vegetables and forages, this process can severely impact quality. Proteases, in particular, are responsible for post-harvest protein degradation and contribute to cellular breakdown. Alfalfa is a high-quality forage and a perennial source of protein for dairy cows. Its nutritional value and suitability for sustainable agricultural production are also driving interest in the possible use of alfalfa for human consumption, however, the issue of rapid protein degradation after harvest needs to be addressed before the protein can be extracted for human use on a large scale. We have optimized a cost-effective, medium-throughput, fluorescence intensity-based protease activity assay specifically for aboveground plant tissue that balances precision and throughput, the latter of which has been a barrier to further study. This method will allow higher-throughput phenotyping of plant protease activity than is currently feasible: one person can process approximately 120 samples per day, compared to approximately 25 with previous methods. We have used this method in alfalfa to assess protease activity during wilting at pH 7.4 and pH 6.3. [ABSTRACT FROM AUTHOR]

S-Palmitoylation is a reversible post-translational modification involving saturated fatty acid palmitate-to-cysteine linkage in the protein, which guides many aspects of macrophage physiology in health and disease. However, the precise role and underlying mechanisms of palmitoylation in Mycobacterium tuberculosis infection of macrophages remain elusive. Here, we found that M. tuberculosis infection induced the expression of zinc-finger DHHC domain-type palmitoyl-transferases (ZDHHCs), particularly ZDHHC2, in mouse macrophages. Furthermore, ZDHHC2 deficiency in mouse macrophages impaired the immunity against M. tuberculosis and reduced the production of various proinflammatory cytokines. Mechanistic studies revealed the involvement of ZDHHC2 in mediating the palmitoylation of B-RAF and C-RAF, affecting their autophagic degradation and stabilizing protein levels. The increased abundance of B-RAF and C-RAF subsequently increases the activity of the extracellular signal-regulated kinase (ERK) signaling pathway, affecting the survival of M. tuberculosis within macrophages. These findings suggest that ZDHHC2 is a potential target for treating tuberculosis. [ABSTRACT FROM AUTHOR]

Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into mitochondria. If the rate of protein synthesis exceeds the capacity of the mitochondrial import machinery, precursor proteins can transiently accumulate in the cytosol. The cytosolic accumulation of mitochondrial precursors jeopardizes cellular protein homeostasis (proteostasis) and can be the cause of diseases. In order to prevent these toxic effects, most non-imported precursors are rapidly degraded by the ubiquitin-proteasome system. However, cells employ a second layer of defense which is the facilitated sequestration of mitochondrial precursor proteins in transient protein aggregates. The formation of such structures is triggered by nucleation factors such as small heat shock proteins. Disaggregases and chaperones can liberate precursors from cytosolic aggregates to pass them on to the mitochondrial import machinery or, under persistent stress conditions, to the proteasome for degradation. Owing to their role as transient buffering systems, these aggregates were referred to as MitoStores. This review articles provides a general overview about the MitoStore concept and the early stages in mitochondrial protein biogenesis in yeast and, in cases where aspects differ, in mammalian cells. [ABSTRACT FROM AUTHOR]

Gram-negative bacterial pathogens inject effector proteins inside plant cells using a type III secretion system. These effectors manipulate plant cellular functions and suppress the plant immune system in order to promote bacterial proliferation. Despite the fact that bacterial effectors are exogenous threatening proteins potentially exposed to the protein degradation systems inside plant cells, effectors are relative stable and able to perform their virulence functions. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, undergoes phosphorylation of specific residues inside plant cells, and this promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. The absence of those specific phosphorylation sites or specific host ubiquitin proteases leads to a substantial decrease in RipE1 protein accumulation, indicating that RipE1 hijacks plant post-translational modification regulators in order to promote its own stability. These results suggest that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens. Author summary: Most bacterial plant pathogens inject effector proteins inside host cells to suppress immune responses and manipulate other plant functions in order to cause disease. Even though these effector proteins could be targeted by the protein degradation systems in plant cells, they are able to perform their virulence functions, which suggests that they are relatively stable, even though the mechanisms leading to this stability remain poorly understood. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, has the potential to be ubiquitinated and degraded in plant cells. However, RipE1 hijacks plant kinases and undergoes phosphorylation of specific residues inside plant cells, and this counteracts its ubiquitination and promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. Our study suggests that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens, and that pathogen effectors hijack plant post-translational modification regulators in order to promote their own stability. [ABSTRACT FROM AUTHOR]

Kiwifruit (Actinidia deliciosa)-derived actinidin, a cysteine protease, is renowned for its meat-tenderizing and milk-clotting activities. Despite its potential in various biotechnological applications, an efficient expression platform for actinidin production has not yet been developed. Instead, actinidin has traditionally been purified directly from the fruits of various plants. This study aimed to produce kiwifruit-derived actinidin in the leaves of Nicotiana benthamiana. The expressed actinidin was directed to the lumen of the endoplasmic reticulum (ER) using the binding immunoglobulin protein (BiP) signal sequence and an ER retention signal. To facilitate cost-effective purification, the family 3 cellulose-binding module (CBM3) was employed as an affinity tag, along with microcrystalline cellulose beads that bind efficiently to CBM3. A significant portion of the expressed actinidin was recovered in the soluble fraction without proteolytic degradation. The purified actinidin exhibited β-casein-degrading activity, with optimal efficiency observed at 55°C and pH 7.0. These results establish a promising plant-based platform for the efficient production and functional application of kiwifruit-derived actinidin in diverse biotechnological processes. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder clinically characterized by progressive decline of memory and cognitive functions, and it is the leading cause of dementia accounting for 60%–80% of dementia patients. A pathological hallmark of AD is the accumulation of aberrant protein/peptide aggregates such as extracellular amyloid plaques containing amyloid-beta peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. These aggregates result from the failure of the proteostasis network, which encompasses protein synthesis, folding, and degradation processes. Autophagy is an intracellular self-digesting system responsible for the degradation of protein aggregates and damaged organelles. Impaired autophagy is observed in most neurodegenerative disorders, indicating the link between autophagy dysfunction and these diseases. A massive accumulation of autophagic vacuoles in neurons in Alzheimer's brains evidences autophagy impairment in AD. Modulating autophagy has been proposed as a therapeutic strategy for AD because of its potential to clear aggregated proteins. However, autophagy modulation therapy for AD is not yet clinically available. This mini-review aims to summarize clinical studies testing potential autophagy modulators for AD and to evaluate their proximity to clinical use. We accessed clinicaltrials.gov provided by the United States National Institutes of Health to identify completed and ongoing clinical trials. Additionally, we discuss the limitations and challenges of these therapies. [ABSTRACT FROM AUTHOR]

Targeted Protein Degradation (TPD) represented by Proteolysis-Targeting Chimeras (PROTAC) is the frontier field in the research and development of antitumor therapy, in which oral drug HP518 Receives FDA Proceed Authorization for its IND Application for Prostate Cancer Treatment. Recently, molecular glue, functioning via degradation of the target protein is emerging as a promising modality for the development of therapeutic agents, while exhibits greater advantages over PROTAC, including improved efficiency, resistance-free properties, and the capacity to selectively target "undruggable" proteins. This marks a revolutionary advancement in the landscape of small molecule drugs. Given that molecular glue research is still in its early stage, we summarized the mechanisms of molecular glue, the promising drugs in clinical trials and diverse feasible design strategies for molecular glue therapeutics. [ABSTRACT FROM AUTHOR]

Simple Summary: Low-protein (LPRO) diets provide benefits such as reduced feed costs and improved utilization. However, the necessary nutritional data for optimizing these diets are incomplete, and formulation techniques are still developing. Moreover, there is no consensus on the impact of low-protein diets with balanced amino acids on production performance. This study examined the impact of protease and Bacillus coagulans on broilers fed LPRO diets, finding that these additives can counteract the negative effects by increasing serum amino acids, upregulating transporter genes, enhancing intestinal barriers, and improving cecal microbiota, offering a basis for promoting LPRO feeds in broiler production. Low-protein (LPRO) diets can effectively reduce feed costs and decrease environmental pollution, making them an important pathway to enhance the sustainability of livestock production. However, they may have adverse effects on the growth performance of broiler chickens, which has limited their widespread application. This study aims to explore the impact of adding protease (PRO) to LPRO diets on the growth performance of broiler chickens, especially under conditions with or without the presence of Bacillus coagulans (BC), in order to provide theoretical support for the scientific application and promotion of LPRO feeds. We selected 432 one-day-old broiler chickens and divided them into four treatment groups, which were fed with the control (CON) diet, the LPRO diet, the PRO diet (LPRO diet with added protease), and the PAB diet (PRO diet with added BC). The LPRO group demonstrated decreased growth performance while both PRO and PAB diets resulted in a significant increase (p < 0.05). Both PRO and PAB diets significantly enhanced the expression of amino acid transport genes and tight junction genes (p < 0.05) and optimized the composition of the intestinal microbiota. Overall, LPRO diets have a detrimental effect on the growth of broiler chickens, while the PRO and PAB diets effectively counteract these negative effects by improving protein digestion, amino acid absorption, and intestinal health. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) are highly contagious pathogens that cause zoonotic disease with limited availability of antiviral therapies, presenting ongoing challenges to both public health and the livestock industry. Unveiling host proteins that are crucial to the IAV life cycle can help clarify mechanisms of viral replication and identify potential targets for developing alternative host-directed therapies. Using a four-dimensional (4D), label-free methodology coupled with bioinformatics analysis, we analyzed the expression patterns of cellular proteins that changed following H9N2 virus infection. Compared to the control group, the H9N2 infected group displayed 732 differentially expressed proteins (DEPs), with 298 proteins showing upregulation and 434 proteins showing downregulation. Gene Ontology (GO) functional analysis showed that DEPs were catalog in 11 biological processes, three cellular components, and eight molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEPs were involved in processes including cytokine signaling pathways induced by virus infection and protein digestion and absorption. Proteins including TP53, DDX58, and STAT3 were among the top hub proteins in the protein–protein interaction (PPI) analysis, suggesting that these signaling cascades could be essential for the propagation of IAVs. Furthermore, the host protein SNAPIN was chosen to ascertain the accuracy of expression changes identified through a proteomic analysis. The results indicated that SNAPIN was downregulated following infection with IAVs both in vitro and in vivo, which is consistent with the proteomics results, suggesting that SNAPIN may serve as a key regulatory factor in the viral life cycle of IAVs. Our research delineates an extensive interaction map of IAV infection within the A549 cells, facilitating the discovery of pivotal proteins that contribute to the virus's propagation, potentially offering target candidates to screen for antiviral therapeutics. [ABSTRACT FROM AUTHOR]