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The RAS family GTPases are the most frequently mutated oncogene family in human cancers. Activating mutations in either of the three RAS isoforms (HRAS, KRAS, or NRAS) are found in nearly 20% of all human tumors with NRAS mutated in ~25% of melanomas. Despite remarkable advancements in therapies targeted against mutant KRAS, NRAS-specific pharmacologics are lacking. Thus, development of inhibitors of NRAS would address a critical unmet need to treating primary tumors harboring NRAS mutations as well as BRAF-mutant melanomas, which frequently develop resistance to clinically approved BRAF inhibitors through NRAS mutation. Building upon our previous studies with the monobody NS1 that recognizes HRAS and KRAS but not NRAS, here we report the development of a monobody that specifically binds to both GDP and GTP-bound states of NRAS and inhibits NRAS-mediated signaling in a mutation-agnostic manner. Further, this monobody can be formatted into a genetically encoded NRAS-specific degrader. Our study highlights the feasibility of developing NRAS selective inhibitors for therapeutic efforts.

The progression and malignancy of many tumors are associated with increased tissue stiffness. Conversely, the oncogenically transformed cells can be confined in soft stroma. Yet, the underlying mechanisms by which soft matrix confines tumorigenesis and metastasis remain elusive. Here, we show that pancreatic cancer cells are suppressed in the soft extracellular matrix, which is associated with YAP1 degradation through autophagic-lysosomal pathway rather than Hippo signal mediated proteasome pathway. In the soft stroma, PTEN is upregulated and activated, which consequently promotes lysosomal biogenesis, leading to the activation of cysteine-cathepsins for YAP1 degradation. In vitro, purified cathepsin L can directly digest YAP1 under acidic conditions. Lysosomal stress, either caused by chloroquine or overexpression of cystatin A/B, results in YAP1 accumulation and malignant transformation. Likewise, liver fibrosis induced stiffness can promote malignant potential in mice. Clinical data show that down-regulation of lysosomal biogenesis is associated with pancreatic fibrosis and stiffness, YAP1 accumulation, and poor prognosis in PDAC patients. Together, our findings suggest that soft stroma triggers lysosomal flux-mediated YAP1 degradation and induces cancer cell dormancy.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and postharvest regulation), plant responses to environmental signals (endoplasmic-reticulum-associated degradation, chloroplast-associated degradation, drought tolerance, and the growth-defense trade-off), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

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]

Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Insulin tightly regulates glucose levels within a narrow range through its action on muscle, adipose tissue and the liver. The activation of insulin receptors activates multiple intracellular pathways with different functions. Another tightly regulated complex system in the body is acid-base balance. Metabolic acidosis, defined as a blood pH < 7.35 and serum bicarbonate < 22 mmol/L, has clear pathophysiologic consequences including an effect on insulin action. With the ongoing intake of typical acid-producing Western diets and the age-related decline in renal function, there is an increase in acid levels within the range considered to be normal. This modest increase in acidosis is referred to as acid stress and it may have some pathophysiological consequences. In this article, we discuss the effects of acid stress on insulin actions in different tissues.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

The proteasome degrades proteins, which is essential for cellular homeostasis. Ubiquitin independent proteolysis degrades highly disordered and misfolded proteins. A decline of proteasomal activity has been associated with multiple neurodegenerative diseases due to the accumulation of misfolded proteins. In this work, cyclic peptide proteasome stimulators (CyPPSs) that enhance the clearance of misfolded proteins were discovered. In the initial screen of predicted natural products (pNPs), several cyclic peptides were found to stimulate the 20S core particle (20S CP). Development of a robust structural activity relationship led to the identification of potent, cell permeable CyPPSs. In vitro assays revealed that CyPPSs stimulate degradation of highly disordered and misfolded proteins without affecting ordered proteins. Furthermore, using a novel flow-based assay for proteasome activity, several CyPPSs were found to stimulate the 20S CP in cellulo. Overall, this work describes the development of CyPPSs as chemical tools capable of stimulating the proteasome and provides strong support for proteasome stimulation as a therapeutic strategy for neurodegenerative diseases.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

Flaviviruses, including Zika virus (ZIKV) and Dengue virus (DENV), rely on their non-structural protein 5 (NS5) for both replication of viral genome and suppression of host IFN signaling. DENV and ZIKV NS5s were shown to facilitate proteosome-mediated protein degradation of human STAT2 (hSTAT2). However, how flavivirus NS5s have evolved for species-specific IFN-suppression remains unclear. Here we report structure-function characterization of the DENV serotype 2 (DENV2) NS5-hSTAT2 complex. The MTase and RdRP domains of DENV2 NS5 form an extended conformation to interact with the coiled-coil and N-terminal domains of hSTAT2, thereby promoting hSTAT2 degradation in cells. Disruption of the extended conformation of DENV2/ZIKV NS5, but not the alternative compact state, impaired their hSTAT2 binding. Our comparative structural analysis of flavivirus NS5s further reveals a conserved protein-interaction platform with subtle amino-acid variations likely underpinning diverse IFN-suppression mechanisms. Together, this study uncovers a conformational selection mechanism underlying species-specific hSTAT2 inhibition by flavivirus NS5.

Proteins and essential fatty acids are crucial components of the human diet. However, lipids and proteins are susceptible to oxidative modification during food processing resulting in changes to their structural characteristics and functional properties. Food products rich in polyunsaturated fatty acids are highly susceptible to lipid peroxidation and generate bifunctional reactive aldehydes. Bifunctional aldehydes such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) readily bind to protein nucleophiles and lead to intra- or intermolecular protein cross-linking. In comparison with lipid oxidation, the degradation of proteins by prooxidants appears to be more intricate and results in a greater diversity of oxidation products. Although individual oxidation processes involving lipids and proteins received increasing attention in the past decades, the interactions between those aldehydes and protein oxidation in food have not been extensively explored. Studies indicate that the reactions of lipid and protein oxidation may take place simultaneously or independently, but oxidation products that arose from one reaction may further interact with lipids or proteins. The present review presents a perspective on reactive aldehydes and the role of aldehydes in inducing protein oxidation in muscle foods. Emphasis is focused on the interaction mechanism of the lipid, protein, and myoglobin protein oxidations. In addition, the occurrence of aldehydes derived from lipid oxidation in food systems as well as the endogenous antioxidant peptides or amino acids in meat and plant proteins are also briefly described. [ABSTRACT FROM AUTHOR]

Cover Legend Illustration of the in planta interactome of ASK1/CUL1, components of the ubiquitin ligase protein complex involved in regulating protein degradation. Ubiquitin molecules are depicted as 3D models, attached to a spherical network surrounding the rosette leaves of Arabidopsis thaliana. Image courtesy of Nitzan Shabek (Sun et al., pp. 2127–2136).. [Extracted from the article]

Background and Purpose: Bifunctional small molecule degraders, which link the target protein with E3 ubiquitin ligase, could lead to the efficient degradation of the target protein. BGB‐16673 is a Bruton's tyrosine kinase (BTK) degrader. A translational PK/PD modelling approach was used to predict the human BTK degradation of BGB‐16673 from preclinical in vitro and in vivo data. Experimental Approach: A simplified mechanistic PK/PD model was used to establish the correlation between the in vitro and in vivo BTK degradation by BGB‐16673 in a mouse model. Human and mouse species differences were compared using the parameters generated from in vitro human or mouse blood, and human or mouse serum spiked TMD‐8 cells. Human PD was then predicted using the simplified mechanistic PK/PD model. Key Results: BGB‐16673 showed potent BTK degradation in mouse whole blood, human whole blood, and TMD‐8 tumour cells in vitro. Furthermore, BGB‐16673 showed BTK degradation in a murine TMD‐8 xenograft model in vivo. The PK/PD model predicted human PD and the observed BTK degradation in clinical studies both showed robust BTK degradation in blood and tumour at clinical dose range. Conclusion and Implications: The presented simplified mechanistic model with reduced number of model parameters is practically easier to be applied to research projects compared with the full mechanistic model. It can be used as a tool to better understand the PK/PD behaviour for targeted protein degraders and increase the confidence when moving to the clinical stage. [ABSTRACT FROM AUTHOR]

Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides. [ABSTRACT FROM AUTHOR]

The physicochemical properties and in vitro digestion behaviours analysis of dairy matrices (liquid cow milk, semi‐liquid yoghurt and semi‐solid cheese) with different fat globules were evaluated. Yoghurt and cheese with large/small‐sized fat globules were produced with raw (~4 μm) and homogenised cow milk (~0.40 μm). During the whole in vitro digestion, small‐sized dairy matrices had a higher lipolysis degree (~70–83%) compared to large‐sized ones (~55–77%) during the whole digestion. Small‐sized dairy matrices showed greater in vitro proteolysis and more porous protein matrix compared to large‐size ones. Dairy matrices with different fat globules exhibited different digestive behaviours, helping design lipid structures in different food matrices and their application in regulating digestive kinetics. [ABSTRACT FROM AUTHOR]

Kallikrein-related peptidase 7 (KLK7) is one of 15 members of the tissue kallikrein family and is primarily expressed in the skin epidermis. The activity of KLK7 is tightly regulated by multiple stages of maturation and reversible inhibition, similar to several other extracellular proteases. In this work, we used protease-specific inhibitors and active site variants to show that KLK7 undergoes autolysis at two separate sites in the 170 and 99 loops (chymotrypsinogen numbering), resulting in a loss of enzymatic activity. A protein BLAST search using the autolyzed KLK7 loop sequences identified mast cell chymase as a potential KLK7 substrate. Indeed, KLK7 cleaves chymase resulting in a concomitant loss of activity. We further demonstrate that KLK7 can hydrolyze other mast cell proteases as well as several cytokines. These cytokines belong mainly to the interferon and IL-10 families including IFN-α, IFN-β, IFN-γ, IL-28A/IFN-λ2, IL-20, IL-22, and IL-27. This is the first study to identify a possible molecular interaction link between KLK7 and mast cell proteases and cytokines. Although the precise biological implications of these findings are unclear, this study extends our understanding of the delicate balance of proteolytic regulation of enzyme activity that maintains physiological homeostasis, and facilitates further biological investigations. [ABSTRACT FROM AUTHOR]

Histone deacetylase (HDAC) enzymes 1–3 exist in several corepressor complexes and are viable drug targets. To date, proteolysis targeting chimeras (PROTACs) designed to target HDAC1–3 typically exhibit the selective degradation of HDAC3. Herein, we report cereblon-recruiting PROTACs that degrade HDAC1 with selectivity over HDAC3. [ABSTRACT FROM AUTHOR]

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules containing a ligand for a protein of interest linked to an E3 ubiquitin ligase ligand that induce protein degradation through E3 recruitment to the target protein. Small changes in PROTAC linkers can have drastic consequences, including loss of degradation activity, but the structural mechanisms governing such changes are unclear. To study this phenomenon, we screened PROTACs of diverse targeting modalities and identified dTAG-13 as an activator of the xenobiotic-sensing pregnane X receptor (PXR), which promiscuously binds various ligands. Characterization of dTAG-13 analogs and precursors revealed interplay between the PXR-binding moiety, linker, and E3 ligand that altered PXR activity without inducing degradation. A crystal structure of PXR ligand binding domain bound to a precursor ligand showed ligand-induced binding pocket distortions and a linker-punctured tunnel to the protein exterior at a region incompatible with E3 complex formation, highlighting the effects of linker environment on PROTAC activity. [Display omitted] • PROTACs can modulate drug metabolism pathways through PXR activation • PROTAC off-target effects extend beyond protein degradation events • The PROTAC dTAG-13 functions as a partial PXR agonist rather than a PXR degrader • The dTAG-13 binding mode is incompatible with PXR-PROTAC-E3 complex formation Huber et al. identify various PROTACs as modulators of PXR activity, highlighting PXR's role as a chemical-sensing regulator of drug metabolism pathways. One such molecule, dTAG-13, is used as a case study to investigate the mechanism of PROTAC-mediated PXR activation, rather than degradation, of PROTAC-bound PXR. [ABSTRACT FROM AUTHOR]

Background: A key factor in the propagation of α-synuclein pathology is the compromised protein quality control system. Variations in membrane association and astrocytic uptake between different α-synuclein forms suggest differences in exocytosis or membrane cleavage, potentially impacting the secretome's influence on dopaminergic neurons. We aimed to understand differences in protein degradation mechanisms of astrocytes for both wild-type (WT) and mutant forms of α-synuclein, specifically during periods of reduced degradation efficiency. We also investigated α-synuclein release into the secretome and its effects on healthy dopaminergic neurons. Methods: Cellular models used were rat primary astrocytes alongside hiPSC-derived astrocytes, whose impact on rat primary dopaminergic neurons and the human SH-SY5Y cell line was investigated. We examined the release and accumulation of α-synuclein resulting from impaired degradatory pathways, including matrix metalloprotease-MMP9, the ubiquitin proteasomal pathway-UPS, and the autophagy-lysosomal pathway-ALP, using immunocytochemical analysis and flow cytometry. Additionally, we explored the effect of astrocytic secretome on dopaminergic-neuronal survival, neurite collapse and function. Results: At early stages, astrocytes were able to deal efficiently with monomeric α-synuclein (via UPS), and larger aggregates (through MMP9 and autophagy), clearing extracellular α-synuclein and maintaining neuronal health. However, extended exposure to extracellular monomeric and aggregated α-synuclein compromised their proteasomal activity, inhibiting MMP9 and destabilizing autophagy, transforming astrocytes from protectors to promoters of neurodegeneration. This study is the first to elucidate the astrocytes' preferred degradation pathways for both monomeric and aggregated forms of α-synuclein, along with the subsequent effects of these payloads on the cellular degradation machinery. The astrocytic transformation is characterized by α-synuclein expulsion, increased release of inflammatory cytokines, and diminished secretion of growth factors leading to dopaminergic neuronal apoptosis and dysfunction, particularly neurite collapse, intracellular Ca2+ response and vesicular dopamine release. The presence of phosphorylated and nitrated α-synuclein species in astrocytes also suggests their potential involvement in modifying both forms of the protein. Conclusion: The initial protective action of astrocytes in clearing and degrading extracellular α-synuclein is severely compromised at latter stages, leading to astrocytic dysfunction and impairing neuron-glia cross-talk. This study underscores the criticality of integrating astrocytes into treatment paradigms in synucleinopathies. [ABSTRACT FROM AUTHOR]

ABSTRACT The progression of periodontitis, a bacteria‐driven inflammatory and bone‐destructive disease, involves myriad cellular and molecular mechanisms. Protein regulation significantly influences the pathogenesis and management of periodontitis. However, research regarding its regulatory role in periodontitis remains relatively limited. The ubiquitin‐proteasome system (UPS), which mainly involves ubiquitination by E3 ubiquitin ligases (E3s) and deubiquitination by deubiquitinating enzymes (DUBs), is the primary intracellular and non‐lysosomal mechanism of protein degradation. Recent studies have provided compelling evidence to support the involvement of UPS in periodontitis progression. Increasing evidence indicated that E3s, such as CUL3, Nedd4‐2, Synoviolin, FBXL19, PDLIM2, TRIMs and TRAFs, modulate inflammatory responses and bone resorption in periodontitis through multiple classical signalling pathways, including NLRP3, GSDMD, NF‐κB, Wnt/β‐catenin and Nrf2. Meanwhile, DUBs, including OTUD1, A20, CYLD, UCH‐L1 and USPs, also broadly modulate periodontitis progression by regulating signalling pathways such as NF‐κB, Wnt/β‐catenin, NLRP3, and BMP2. Therefore, the modulation of E3s and DUBs has proven to be an effective therapy against periodontitis. This review provides a comprehensive overview of the regulatory role of ubiquitinating and deubiquitinating enzymes in periodontitis progression and the underlying mechanisms. Finally, we summarise several chemical and genetic methods that regulate UPS enzymes and pave the way for the development of targeted therapies for periodontitis. [ABSTRACT FROM AUTHOR]

Elevated neutrophil counts in broncho-alveolar lavage (BAL) fluids of lung transplant (LTx) patients with chronic lung allograft dysfunction (CLAD) are associated with disease pathology. However, phenotypical characteristics of these cells remained largely unknown. Moreover, despite enhanced levels of the most potent human neutrophil-attracting chemokine CXCL8 in BAL fluid, no discrimination had been made between natural NH2-terminally truncated CXCL8 proteoforms, which exhibit up to 30-fold differences in biological activity. Therefore, we aimed to characterize the neutrophil maturation and activation state, as well as proteolytic activation of CXCL8, in BAL fluids and peripheral blood of LTx patients with CLAD or infection and stable LTx recipients. Flow cytometry and microscopy revealed a high diversity in neutrophil maturity in blood and BAL fluid, ranging from immature band to hypersegmented aged cells. In contrast, the activation phenotype of neutrophils in BAL fluid was remarkably homogeneous. The highly potentiated NH2-terminally truncated proteoforms CXCL8(6–77), CXCL8(8–77) and CXCL8(9–77), but also the partially inactivated CXCL8(10–77), were detected in BAL fluids of CLAD and infected LTx patients, as well as in COVID-19 and influenza patient cohorts by tandem mass spectrometry. Moreover, the most potent proteoform CXCL8(9–77) specifically correlated with the neutrophil counts in the LTx BAL fluids. Finally, rapid proteolysis of CXCL8 in BAL fluids could be inhibited by a combination of serine and metalloprotease inhibitors. In conclusion, proteolytic activation of CXCL8 promotes neutrophilic inflammation in LTx patients. Therefore, application of protease inhibitors may hold pharmacological promise for reducing excessive neutrophil-mediated inflammation and collateral tissue damage in the lungs. [ABSTRACT FROM AUTHOR]

ABSTRACT Drug‐resistant tuberculosis infections are a major threat to global public health. The essential mycobacterial ClpC1P1P2 protease has received attention as a prospective target for novel antibacterial therapeutics. However, efforts to probe its function in cells are constrained by our limited knowledge of its physiological proteolytic repertoire. Here, we interrogate the role of mycobacterial ClpS in directing N‐degron pathway proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis. Binding assays demonstrate that mycobacterial ClpS binds canonical primary destabilizing residues (Leu, Phe, Tyr, Trp) with moderate affinity. N‐degron binding restricts the conformational flexibility of a loop adjacent to the ClpS N‐degron binding pocket and strengthens ClpS•ClpC1 binding affinity ~30‐fold, providing a mechanism for cells to prioritize N‐degron proteolysis when substrates are abundant. Proteolytic reporter assays in M. smegmatis confirm degradation of substrates bearing primary N‐degrons, but suggest that secondary N‐degrons are absent in mycobacteria. This work expands our understanding of the mycobacterial N‐degron pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors. [ABSTRACT FROM AUTHOR]

Redox-mediated posttranslational modification, as exemplified by protein S-nitrosylation, modulates protein activity and function in both health and disease. Here, we review recent findings that show how normal aging, infection/inflammation, trauma, environmental toxins, and diseases associated with protein aggregation can each trigger excessive nitrosative stress, resulting in aberrant protein S-nitrosylation and hence dysfunctional protein networks. These redox reactions contribute to the etiology of multiple neurodegenerative disorders as well as systemic diseases. In the CNS, aberrant S-nitrosylation reactions of single proteins or, in many cases, interconnected networks of proteins lead to dysfunctional pathways affecting endoplasmic reticulum (ER) stress, inflammatory signaling, autophagy/mitophagy, the ubiquitin-proteasome system, transcriptional and enzymatic machinery, and mitochondrial metabolism. Aberrant protein S-nitrosylation and transnitrosylation (transfer of nitric oxide [NO]-related species from one protein to another) trigger protein aggregation, neuronal bioenergetic compromise, and microglial phagocytosis, all of which contribute to the synapse loss that underlies cognitive decline in Alzheimer's disease and related dementias. In this review, Oh, Nakamura, Zhang, and Lipton discuss diverse mechanisms of synaptic loss mediated by aberrant protein S-nitrosylation in neurodegenerative disorders. Mechanisms affected include ER stress, inflammatory signaling, autophagy/mitophagy, the ubiquitin-proteasome system, transcriptional and enzymatic machinery, and mitochondrial metabolism. [ABSTRACT FROM AUTHOR]

Targeted protein degradation (TPD) has emerged as an important strategy to target disease-relevant proteins that were previously considered difficult to drug or even undruggable. Cereblon (CRBN) plays an outsized role in TPD as a preferred degradation-inducing effector protein for several reasons, including its anticipated broad protein substrate scope and its ligandability with drug-like small molecules. Notably, CRBN-based molecular glue degraders (MGDs) and proteolysis targeting chimeras (PROTACs) have shown success in clinical trials and, in some cases, as approved drugs. Thus, the interest in CRBN ligands within the pharmaceutical industry and academia has increased dramatically in recent years, highlighting the need for robust synthetic approaches towards them. This short review summarizes tactics and strategies to synthesize CRBN ligands, including the most recent developments in the field. Particular emphasis is put on the construction and direct functionalization of key CRBN binding motifs such as glutarimides and dihydrouracils. 1 Introduction 2 Cereblon Ligands with Glutarimide Binding Motif 3 Cereblon Ligands with Dihydrouracil Binding Motif 4 Cereblon Ligands with Other Binding Motifs 5 Conclusions and Outlook [ABSTRACT FROM AUTHOR]

Background: The Japanese icefish (Salangichthys microdon) is a valuable food resource in Japan; however, its freshness rapidly diminishes during transport and cold storage. In this study, the freshness change during chilled (5°C) and super‐chilled (−1 and −2°C) of S. microdon samples collected from Kasumigaura lake in October and December was assessed by observing their surface structure and determining their drip loss, K values, and free amino acid content. Results: The results revealed that more wrinkles formed on the surface of S. microdon samples with increasing storage time. More drip loss occurred in fall than in winter. Higher K values and increased degradation of muscle proteins were observed in winter. Moreover, their free amino acid content increased with storage time at all temperatures. Conclusion: This study suggested that seasonal differences and storage temperature should be considered in selecting the appropriate fishing, handling, and storage methods to maintain the freshness and quality of S. microdon. [ABSTRACT FROM AUTHOR]

We illustrate and discuss the relevance of key endoplasmic reticulum (ER)-associated RNA decay pathways – including inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), RIDD lacking endomotif (RIDDLE), ER-associated RNA silencing (ERAS), regulation of aberrant protein production (RAPP), and ER nonsense-mediated mRNA decay (ER-NMD) – for the unfolded protein response (UPR) and ER homeostasis. Multiple parallel ER-associated RNA decay pathways may exist to ensure substrate specificity and to efficiently address diverse ER homeostasis and viral challenges. We summarize recent insights into the intricate interplay of these pathways, and highlight gaps in our knowledge regarding target selectivity, recruitment of degradation machinery, and the physiological relevance of ER-associated RNA decay pathways. An emerging ER–ribosome-associated quality control (RQC) mechanism involves coordinated ER-associated processes of RNA degradation – IRE1α, Argonaute 2 (AGO2), UPF1 – and protein degradation (LTN1, HRD1, VCP). The endoplasmic reticulum (ER) is central to the processing of luminal, transmembrane, and secretory proteins, and maintaining a functional ER is essential for organismal physiology and health. Increased protein-folding load on the ER causes ER stress, which activates quality control mechanisms to restore ER function and protein homeostasis. Beyond protein quality control, mRNA decay pathways have emerged as potent ER fidelity regulators, but their mechanistic roles in ER quality control and their interrelationships remain incompletely understood. Herein, we review ER-associated RNA decay pathways – including regulated inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), nonsense-mediated mRNA decay (NMD), and Argonaute-dependent RNA silencing – in ER homeostasis, and highlight the intricate coordination of ER-targeted RNA and protein decay mechanisms and their association with antiviral defense. [ABSTRACT FROM AUTHOR]

This study investigated the kinetics of ultrasound (US)-assisted hydrolysis of pequi almond protein (PAP) and the conformational and multifunctional characteristics of the hydrolysates. The US-assisted enzymolysis of PAP using Alcalase® was conducted in a US bath (25 kHz, 38 W/L) for 180 min at 60 °C and pH 7.5. The results showed an increase in the rate of proteolysis by 11.3 %, leading to a higher final degree of hydrolysis (25.2 %) and a greater concentration of TCA-soluble proteins after 45 min and 180 min (29 % and 8 %, respectively). Both conventional and US-assisted hydrolysis led to the breakdown of the protein structure, forming low molecular weight peptides (<15 kDa). However, US-assisted hydrolysis demonstrated greater fragmentation of the PAP surface, enhancing enzymatic efficacy. Intrinsic fluorescence and UV spectroscopy indicated increased exposure of hydrophobic groups throughout hydrolysis, and FTIR spectroscopy revealed changes in the amide bands, suggesting structural transitions in the protein. Finally, the hydrolysates obtained from the USassisted reaction showed higher solubility (≤27 % at pH 6.0), foaming capacity (≤82 % at pH 6.0), and antioxidant activity (≤40 %). Therefore, these results indicate the potential of the US to improve the performance of Alcalase® in the hydrolysis of PAP, producing compounds of industrial interest. [Display omitted] • Ultrasound (US)-assisted hydrolysis of Pequi almond protein (PAP) was first studied. • US enhanced PAP hydrolysis, increasing the rate (11.3 %) and peptide production (8 %). • Hydrolysis and US increased the Pequi almond protein solubility (<2.6x at pH 4.0). • US-assisted hydrolysis increased the foaming capacity of PAP (<4.4x at pH 6.0). • US-assisted hydrolysis generated higher antioxidant activity peptides (<40 %). [ABSTRACT FROM AUTHOR]

Methyl thiophanate (MT) is a widely used fungicide in agriculture; however, it presents substantial health risks to humans. Exposure to MT can damage the human nervous and reproductive systems and inhibit pepsin, a crucial enzyme for digestion and metabolism. This research aimed to investigate the interaction between MT and pepsin using different spectroscopic techniques and dynamic simulations. The UV-Vis results further validated the Trp delocalization findings through molecular docking. At various temperature levels, the effective quenching constants (k q) were found to be 8.28×103, 5.36×103 and 2.43×103 L mol−1, respectively, indicating static quenching with a k q greater than 2 × 1010. Thermodynamic analysis revealed Δ H 0 = −56.56 kJ mol − 1 and ∆ S 0 = −102 J mol − 1 K − 1 , highlighting the significance of hydrogen bonds and Van der Waals interactions in the complex, consistent with docking analyses. By studying the interaction between pepsin and MT, valuable insights can be gained into its impact on digestive system enzymes, leading to a better understanding of MT's toxicological and inhibitory effects on digestion. This interaction disrupts protein digestion, which has broader implications for nutrient absorption and overall digestive health. [Display omitted] • Pesticides are essential agricultural chemicals to kill harmful organisms. • The conformational of pepsin alter after binding to Methyl Thiophanate. • Structural changes lead to changes in pepsin function. • The results obtained in this in vitro and in silico confirm another. [ABSTRACT FROM AUTHOR]

As a carnivorous fish, the cannibalism are prone to happen in the early seedling stage in Dongting catfish (Silurus asotus), especailly when there is not enough food, which resulting in the low survival rate of Dongting catfish. To find clues to improve the survival rate of Dongting catfish offspring, the comparative transcriptomics analysis were conducted between normal and malformed larval fish, as well as normal larval fish before and after first food intake. In normal and malformed larval fish groups, a total of 881 differential genes were identified, including 634 up-regulated genes and 247 down-regulated genes. From these genes related to growth hormone synthesis and secretion were screened out as SLA isoform X1, SOCS3, STAT1b, JUNB, respectively. In the group of larval fish before and after the first food intake, 4901 up-regulated genes and 3660 down-regulated genes were found, from which the differential genes related to protein digestion and absorption pathway and pancreatic secretion pathway were screened out as cathepsin D precursor, elastase 2 like precursor, elastase 2 precursor, LPL isoform X1, CTRB1 precursor, CTRL precursor, CPA precursor, FABP, SLC15A1, CEL tandem duplicate 2 precursor. The differential genes screened for association with amino acid metabolism were ASAOC precursor, PLD3, NDPKs, trehalase, UMP-CMP kinase, L-amino-acid oxidase, PLB1, FALDH isoform X2, PLA2 precursor, glucokinase. Mean while, nine differential genes were selected for qPCR verification. The results confirmed that the relative expression trends of these genes were consistent with those found in the transcriptome. These findings provide an important reference for developing strategies to enhance the survival rate of Dongting catfish at early seedling stage. [ABSTRACT FROM AUTHOR]

This study explored the nutritional qualities of black mash bean flour impacted by fermentation, gelatinization and their combination. Protease activity, in vitro protein digestibility (IVPD), and protein solubility were evaluated for both fermented and unfermented black mash bean flour samples. Interestingly, when compared to fermented samples and gelatinized mash bean flour (GMBF), raw mash bean flour (RMBF) showed the highest protein solubility. Due to enzymatic activity and protein hydrolysis, fermentation with Lactobacillus E14 and Saccharomyces cerevisiae MK-157 enhanced protein solubility. Prolonged fermentation timeframes were positively correlated with protease activity and IVPD, suggesting the degradation of complex proteins. The highest levels of IVPD (90.12%) and protease activity (5546.78 U/g) were detected in GMBF fermented by S. cerevisiae MK-157, indicating the impact of fermentation on protein breakdown. The Lactobacillus E14-fermented RMBF exhibited 5521.45 U/g of protease activity and 87.54% IVPD. The mineral content of the fermented samples was substantially greater than that of the unfermented GMBF. The increased calcium, magnesium, potassium, sodium, iron, zinc, and copper levels for the GMBF fermented by S. cerevisiae MK-157 indicated that fermentation facilitated the release of minerals from chelated compounds. Microscopic examination revealed higher protein quantities in the fermented flour as well as hydrolyzed and broken starch granules embedded in the protein matrix. Large mash bean proteins were shown to be proteolyzed during fermentation into smaller peptides by SDS PAGE. Storage proteins were extensively broken down during fermentation with S. cerevisiae MK-157, whereas Lactobacillus E14 displayed a more focused pattern of hydrolysis. The nutritional profile of black mash bean flour was improved by fermentation with Lactobacillus E14 and S. cerevisiae MK-157. [ABSTRACT FROM AUTHOR]

Summary: Most dietary fibres improved rheological properties but negatively impacted digestibility. However, the effect of psyllium husk powder (PHP) on digestibility has not been yet reported. Thus, this study aimed to evaluate the rheological properties and digestibility of chicken myofibrillar protein (MP) containing different concentrations of PHP (0%, 0.5%, 1.0%, 1.5%) at 0.45 m NaCl. To assess the rheological properties of MP, viscosity, hydrophobicity, disulphide bond, SDS–PAGE, cooking yield and gel strength were evaluated. The addition of PHP enhanced water retention capacity and protein–protein interactions in the MP system. To evaluate the effect of PHP on digestibility, protein digestibility, SDS–PAGE and microstructure observations were performed. It was confirmed that the addition of PHP improved the protein digestibility of MP by measuring the differences in protein content (%). Therefore, the addition of PHP into MP gel improved not only the rheological properties but also the protein digestibility during in vitro digestion. [ABSTRACT FROM AUTHOR]

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) possess analgesic and anti-inflammatory properties by inhibiting cyclooxygenase (COX) enzymes. Conflicting evidence exists on whether NSAIDs influence signaling related to muscle adaptations and exercise with some research finding a reduction in muscle protein synthesis signaling via the AKT-mTOR pathway, changes in satellite cell signaling, reductions in muscle protein degradation, and reductions in cell proliferation. In this study, we determined if a single maximal dose of flurbiprofen (FLU), celecoxib (CEL), ibuprofen (IBU), or a placebo (PLA) affects the short-term muscle signaling responses to plyometric exercise. Methods: This was a block randomized, double-masked, crossover design, where 12 participants performed four plyometric exercise bouts consisting of 10 sets of 10 plyometric jumps at 40% 1RM. Two hours before exercise, participants consumed a single dose of celecoxib (CEL 200 mg), IBU (800 mg), FLU (100 mg) or PLA with food. Muscle biopsy samples were collected before and 3-h after exercise from the vastus lateralis. Data were analyzed using a repeated measures (RM) ANOVA, ANOVA, or a Friedman test. Significance was considered at p < 0.05. Results: We found no treatment effects on the mRNA expression of PTSG1, PTSG2, MYC, TBP, RPLOP, MYOD1, Pax7, MYOG, Atrogin-1, or MURF1 (all, p > 0.05). We also found no treatment effects on AKT-mTOR signaling or MAPK signaling measured through the phosphorylation status of mTORS2441, mTORS2448, RPS6 235/236, RPS 240/244, 4EBP1, ERK1/2, p38 T180/182 normalized to their respective total abundance (all, p > 0.05). However, we did find a significant difference between MNK1 T197/202 in PLA compared to FLU (p <.05). Conclusion: A single, maximal dose of IBU, CEL, or FLU taken prior to exercise did not affect the signaling of muscle protein synthesis, protein degradation, or ribosome biogenesis three hours after a plyometric training bout. [ABSTRACT FROM AUTHOR]

Protein degradation is a common occurrence in tilapia during the refrigeration process, leading to the generation of unpleasant odor and impacting overall flavor and quality. This study characterized the protein degradation of tilapia during cold storage and utilized amino acids metabolomics analysis to identify metabolic pathways and differential metabolites associated with odor formation and changes. The protein degradation of tilapia during cold storage was characterized through analysis of trichloroacetic acid (TCA) soluble peptides, protein concentration, electrophoresis patterns, protein secondary and tertiary structures, as well as microscopic structures. The characterization of these indicators revealed an obvious protein degradation and structural disruption of tilapia during cold storage. Changes in the types and contents of free amino acids (FAAs) were discussed. Protein degradation produced a large number of FAAs, with Lys contributing the most to flavor changes. By employing amino acids metabolomics analysis, primary and secondary differential metabolites were selected, along with differential metabolic pathways based on amino acids metabolism. And the related amino acid metabolic network was constructed. This study contributes to revealing the impact of amino acids metabolism on odor formation and changes, providing further insights for the research and regulation of odor. [ABSTRACT FROM AUTHOR]

BACKGROUND: The gelation properties of surimi gel under various high temperatures (115, 118, and 121 °C) and sterilization intensities (F0 values of 3–7 min) were systematically investigated. A kinetic model detailed quality changes during heat treatment through mathematical analysis, elucidating mechanisms for gel quality degradation. RESULTS: Increased sterilization intensity significantly reduced the quality characteristics of surimi gel. Compared to the gel without sterilization treatment, when the sterilization intensity was increased to 7 min, the gel strength of the groups treated at 115 °C, 118 °C, and 121 °C decreased by 68.35%, 51.4%, and 51.71%, respectively, and the water‐holding capacity decreased by 24.87%, 16.85%, and 22.5%, respectively. The hardness, chewiness, and whiteness of the gel also significantly decreased, and the changes in these indicators all conformed to a first‐order kinetic model. Activation energy of 291.52 kJ mol−1 highlighted gel strength as the least heat‐resistant. At equivalent sterilization intensities, 115 °C exhibited the poorest gel quality, followed by 121 °C, with 118 °C showing relatively better gel quality. Increased T22 and decreased PT22 suggested heightened water mobility and transition of immobilized water within the gel into free water. Protein degradation, weakened disulfide bonds and hydrophobic interaction, and protein conformation changes collectively led to a rough and incoherent gel network structure with large fissures, as verified by the results of scanning electron microscopy. Correlation analysis indicated potential for precise control over surimi gel quality by modulating physicochemical attributes. CONCLUSION: The outcomes may be beneficial to improve the production and quality control of ready‐to‐eat surimi‐based products. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

CASK (MIM#300172), encoding a calcium/calmodulin‐dependent serine protein kinase, is crucial for synaptic transmission and gene regulation during neural development. Pathogenic variants of CASK are known to cause several neurodevelopmental disorders, including X‐linked intellectual disability and microcephaly with pontine and cerebellar hypoplasia (MICPCH). This study introduces a novel, de novo synonymous CASK variant (NM_001367721.1: c.1737G>A, p.(Glu579=)), discovered in a male patient diagnosed with MICPCH, characterized by microcephaly, developmental delay, visual impairment, and myoclonic seizures. The variant disrupts a donor splice‐site at the end of exon 18. Transcriptomic analysis of blood identified 12 different CASK transcripts secondary to the synonymous variant. Nearly one third of these transcripts were predicted to result in nonsense mediated decay or protein degradation. Protein modeling revealed structural alterations in the PDZ functional domain of CASK, due to exon 18 deletion. Our findings highlight the utility of transcriptomic analysis in demonstrating the underlying disease mechanism in neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Oocytes and early embryos are exposed to many uncontrollable factors that trigger endoplasmic reticulum (ER) stress during in vitro culture. Prevention of ER stress is an effective way to improve the oocyte maturation rate and oocyte quality. Increasing evidence suggests that dietary intake of sufficient n-3 polyunsaturated fatty acids (PUFAs) is associated with health benefits, particularly in the domain of female reproductive health. We found that supplementation of eicosatrienoic acid (ETA) during in vitro maturation (IVM) of oocyte significantly downregulated ER stress-related genes. Mitochondria-associated membranes (MAMs) are communications areas between the ER and mitochondria. Inositol 1,4,5-trisphosphate receptor (IP3R) is a key calcium channels in MAMs and, participates in the regulation of many cellular functions. Notably, the MAM area was significantly decreased in ETA-treated oocytes. CDGSH iron sulfur domain 2 (CISD2) is presents in MAMs, but its role in oocytes is unknown. ETA treatment significantly increased CISD2 expression, and siRNA-mediated knockdown of CISD2 blocked the inhibitory effect of ETA on IP3R. Transcriptomic sequencing and immunoprecipitation experiments showed that ETA treatment significantly decreased expression of the E3 ubiquitin ligase PRKN. PRKN induced ubiquitination and degradation of CISD2, indicating that the PRKN-mediated ubiquitin-proteasome system regulates CISD2. In conclusion, our study reveals the mechanism by which ETA supplementation during IVM alleviates mitochondrial calcium overload under ER stress conditions by decreasing PRKN-mediated ubiquitination of CISD2 and facilitating inhibition of IP3R by CISD2/BCL-2. This improves oocyte quality and subsequent embryo developmental competence prior to implantation. [Display omitted] • Supplementation with ETA improves the quality of oocytes in vitro culture. • ETA regulates calcium homeostasis through PRKN-mediated ubiquitination of CISD2. • CISD2 agonists enhance oocyte and subsequent embryo developmental competence. [ABSTRACT FROM AUTHOR]

During embryonic development, vitamins are involved in energy metabolism and protein synthesis, while cathepsins and antioxidant defense play key roles in yolk degradation and protecting fish embryos from oxidative damage. This study aimed to investigate the consumption of vitamins (A, B1, B2, C, D, and E), ontogenesis of cathepsins, and antioxidant capacity in developing embryos of turbot. In this study, embryos were collected over time from 2.3 to 102.5 h (i.e., hatching) after fertilization for vitamins, cathepsins, and antioxidant capacity analyses. Results showed that the embryonic growth rate in the cleavage stage (CS) was faster than in other stages, while that in the segmentation stage (SS) was lower than in other stages. Embryos displayed higher demands on VA during blastula stage (BS), gastrula stage (GS), the early SS, and hatching day (HD), VB1 at CS; VB2 at BS, GS, SS, and HD; VD at CS and SS; VE at CS, BS, and the late SS; and Vc during entire periods. Cathepsins A (CA) showed a high level at early SS, suggesting its involvement in yolk degradation. Cathepsins B (CB) and cathepsins D (CD) were highly active at GS, suggesting an active apoptosis process. CA, CB, CD, and cathepsins L (CL) all kept low levels in CS, revealing low protein demand during the cleavage process. On the whole, the predominant antioxidant enzymes were superoxide dismutase (SOD) and malondialdehyde (MDA) from CS to SS; SOD, MDA, glutathione peroxidase (GSH-Px), and catalase (CAT) during SS; and GSH-Px and CAT during HD. In conclusion, the fastest growth rate and the slowest growth rate of embryos occurred in CS and SS, respectively, being consistent with energy allocation. Vitamin consumption and cathepsin ontogenesis were stage-specific. Oxidative damage on embryos was relatively less at CS, but larger at SS and HD. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc). Results: The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35 mg/kg leaf fresh weight). Conclusions: Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics. [ABSTRACT FROM AUTHOR]

Evaluating protein and mitochondrial alterations post‐mortem can contribute to determining correlations between fish‐processing parameters and ultimate fish muscle quality. The myofibrillar protein alteration during rigor mortis directly affects the texture of fish muscle. To identify the mechanisms behind post‐mortem softness and quality deterioration, it is crucial to understand the conditions linked to the breakdown of myofibrillar proteins in fish skeletal muscle. Therefore, monitoring protein breakdown at the molecular level and finding target proteins would be considered a marker for fish freshness. Mitochondria play an important role in executing and regulating cell death processes, including apoptosis and necrosis. The mitochondria are the seat of cellular respiration and experience significant alterations in post‐mortem tissues. Processes used to reduce protein degradation, such as optimizing chilling and handling practices, would also minimize mitochondrial changes in fillet quality. Moreover, pH fluctuations are considered a critical point that influences both protein and mitochondrial changes. This review considered the implications of protein and mitochondrial alteration during post‐mortem storage in fish fillets and the possible pathways of their interaction on fillet quality. Mitochondrial characteristics, such as membrane integrity, pH, and ATP levels, are important for post‐mortem muscle cell changes, serving as an early indicator of fish freshness. Understanding the mechanisms behind protein degradation in fish muscle led to maintaining fillet quality and requires further experiments. Label‐free proteomics combined with bioinformatics is crucial for comprehending protein degradation mechanisms to provide customers with safe and fresh fish products while minimizing economic losses associated with fillet deterioration. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Viruses encode strategies to degrade cellular proteins to promote infection and pathogenesis. Here, we revealed that the non-structural protein NSs of Rift Valley fever virus forms a filamentous E3 ligase to trigger efficient degradation of targeted proteins. Reconstitution in vitro and cryoelectron microscopy analysis with the 2.9-Å resolution revealed that NSs forms right-handed helical fibrils. The NSs filamentous oligomers associate with the cellular FBXO3 to form a remodeled E3 ligase. The NSs-FBXO3 E3 ligase targets the cellular TFIIH complex through the NSs-P62 interaction, leading to ubiquitination and proteasome-dependent degradation of the TFIIH complex. NSs-FBXO3-triggered TFIIH complex degradation resulted in robust inhibition of antiviral immunity and promoted viral pathogenesis in vivo. Furthermore, it is demonstrated that NSs can be programmed to target additional proteins for proteasome-dependent degradation, serving as a versatile targeted protein degrader. These results showed that a virulence factor forms a filamentous and programmable degradation machinery to induce organized degradation of cellular proteins to promote viral infection. [Display omitted] • The RVFV NSs protein forms a helical filament • The NSs filament associates with FBXO3 to form a filamentous E3 ligase • The NSs-FBXO3 filamentous E3 ligase mediates proteasome degradation of TFIIH complex • The NSs filament structure can be programmed to target other proteins for degradation The non-structural protein NSs of Rift Valley fever virus forms a filamentous E3 ligase to trigger efficient degradation of the cellular TFIIH complex, leading to robust inhibition of antiviral immunity and enhanced viral pathogenesis. The NSs filament structure can be programmed to target other proteins for proteasome-dependent degradation, serving as a versatile targeted protein degrader. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC), characterized by a high incidence rate, poses significant clinical challenges owing to its poor prognosis despite advancements in diagnostic and therapeutic approaches. Therefore, a comprehensive understanding of the molecular mechanisms driving GC progression is crucial for identifying predictive markers and defining treatment targets. Deubiquitinating enzymes (DUBs), also called deubiquitinases, function as reverse transcriptases within the ubiquitin-proteasome system to counteract protein degradation. Recent findings suggest that DUB dysregulation could be a crucial factor in GC pathogenesis. In this review, we examined recent research findings on DUBs in the context of GC, elucidating their molecular characteristics, categorizations, and roles while also exploring the potential mechanisms underlying their dysregulation in GC. Furthermore, we assessed the therapeutic efficacy of DUB inhibitors in treating malignancies and evaluated the prevalence of aberrant DUB expression in GC. [ABSTRACT FROM AUTHOR]

Background: Cellular iron metabolism is essential for maintaining various biological processes in organisms, and this is influenced by the function of iron-responsive element-binding protein 2 (IRP2), encoded by the IREB2 gene. Since 2019, three cases of a genetic neurodegenerative syndrome resulting from compound heterozygous mutations in IREB2 have been documented, highlighting the crucial role of IRP2 in regulating iron metabolism homeostasis. This study aims to investigate the molecular basis in a single proband born to non-consanguineous healthy parents, presenting with severe psychomotor developmental abnormalities and microcytic anemia. Methods: Trio-whole exome sequencing (WES) was applied to identify the disease-causing gene in an 8-month-old male patient from China. In silico tools were used to predict the pathogenicity of the identified variants, and in vitro functional studies were performed to evaluate the molecular mechanism. Results: WES identified novel biallelic variants, c.1111 A > G (P.Ile371Val) and c.2477 A > T (P.Asp826Val), in the IREB2 gene, which encodes the iron metabolism-related protein, IRP2. Functional studies revealed that c.2477 A > T resulted in a significant degradation of IRP2, which led to the misregulation of intracellular ferric iron. Conclusions: We report the identification of the first functional domain associated with the degradation of IRP2. The biallelic variants that affect protein degradation likely underlie the pathogenesis of the IRP2-related neurodegenerative disorder. Moreover, the use of proteasome inhibitors can potentially restore the expression of IRP2, highlighting a promising therapeutic target for patients with IRP2deficiency. [ABSTRACT FROM AUTHOR]

Background: Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods: Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines, induced pluripotent stem cell-derived dopaminergic neurons and midbrain organoids and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results: We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions: These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein. [ABSTRACT FROM AUTHOR]

ABSTRACT Targeted protein degradation (TPD) has emerged as a highly promising approach for eliminating disease‐associated proteins in the field of drug discovery. Among the most advanced TPD technologies, PROteolysis TArgeting Chimera (PROTAC), functions by bringing a protein of interest (POI) into proximity with an E3 ubiquitin ligase, leading to ubiquitin (Ub)‐dependent proteasomal degradation. However, the designs of most PROTACs are based on the utilization of a limited number of available E3 ligases, which significantly restricts their potential. Recent studies have shown that phytoplasmas, a group of bacterial plant pathogens, have developed several E3‐ and ubiquitin‐independent proteasomal degradation (UbInPD) mechanisms for breaking down host targets. This suggests an alternative approach for substrate recruitment and TPD. Here, we present existing evidence that supports the feasibility of UbInPD in eukaryotic cells and propose candidate proteins that can serve as docking sites for the development of E3‐independent PROTACs. [ABSTRACT FROM AUTHOR]