Pro-inflammatory cytokines in muscle play a pivotal role in physiological responses and in the pathophysiology of inflammatory disease and muscle atrophy. Lactobacillus delbrueckii (LD), as a kind of probiotics, has inhibitory effects on pro-inflammatory cytokines associated with various inflammatory diseases. This study was conducted to explore the effect of dietary LD on the lipopolysaccharide (LPS)-induced muscle inflammation and atrophy in piglets and to elucidate the underlying mechanism. A total of 36 weaned piglets (Duroc × Landrace × Large Yorkshire) were allotted into three groups with six replicates (pens) of two piglets: (1) Nonchallenged control; (2) LPS-challenged (LPS); (3) 0.2% LD diet and LPS-challenged (LD+LPS). On d 29, the piglets were injected intraperitoneally with LPS or sterilized saline, respectively. All piglets were slaughtered at 4 h after LPS or saline injection, the blood and muscle samples were collected for further analysis. Our results showed that dietary supplementation of LD significantly attenuated LPS-induced production of pro-inflammatory cytokines IL-6 and TNF-α in both serum and muscle of the piglets. Concomitantly, pretreating the piglets with LD also clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the muscle, which correlated with the anti-inflammatory effects of LD on the muscle of piglets. Meanwhile, LPS-induced muscle atrophy, indicated by a higher expression of muscle atrophy F-box, muscle RING finger protein (MuRF1), forkhead box O 1, and autophagy-related protein 5 (ATG5) at the transcriptional level, whereas pretreatment with LD led to inhibition of these upregulations, particularly genes for MuRF1 and ATG5. Moreover, LPS-induced mRNA expression of endoplasmic reticulum stress markers, such as eukaryotic translational initiation factor 2α (eIF-2α) was suppressed by pretreatment with LD, which was accompanied by a decrease in the protein expression levels of IRE1α and GRP78. Additionally, LD significantly prevented muscle cell apoptotic death induced by LPS. Taken together, our data indicate that the anti-inflammatory effect of LD supply on muscle atrophy of piglets could be likely regulated by inhibiting the secretion of pro-inflammatory cytokines through the inactivation of the ER stress/NF-κB singling pathway, along with the reduction in protein degradation.

UNASSIGNED: Defective ribosomal products (DRiPs) are non-functional proteins rapidly degraded during or after translation being an essential source for MHC class I ligands. DRiPs are characterized to derive from a substantial subset of nascent gene products that degrade more rapidly than their corresponding native retiree pool. So far, mass spectrometry analysis revealed that a large number of HLA class I peptides derive from DRiPs. However, a specific viral DRiP on protein level was not described. In this study, we aimed to characterize and identify DRiPs derived from a viral protein.
UNASSIGNED: Using the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) which is conjugated N-terminally to ubiquitin, or the ubiquitin-like modifiers FAT10 or ISG15 the occurrence of DRiPs was studied. The formation and degradation of DRiPs was monitored by western blot with the help of a FLAG tag. Flow cytometry and cytotoxic T cells were used to study antigen presentation.
UNASSIGNED: We identified several short lived DRiPs derived from LCMV-NP. Of note, these DRiPs could only be observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, but not in the wild type form. Using proteasome inhibitors, we could show that degradation of LCMV-NP derived DRiPs were proteasome dependent. Interestingly, the synthesis of DRiPs could be enhanced when cells were stressed with the help of FCS starvation. An enhanced NP118-126 presentation was observed when the LCMV-NP was modified with ubiquitin or ubiquitin-like modifiers, or under FCS starvation.
UNASSIGNED: Taken together, we visualize for the first time DRiPs derived from a viral protein. Furthermore, DRiPs formation, and therefore MHC-I presentation, is enhanced under cellular stress conditions. Our investigations on DRiPs in MHC class I antigen presentation open up new approaches for the development of vaccination strategies.

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.

UNASSIGNED: Bromodomain-containing protein 4 (BRD4), an important epigenetic reader, is closely associated with the pathogenesis and development of many diseases, including various cancers, inflammation, and infectious diseases. Targeting BRD4 inhibition or protein elimination with small molecules represents a promising therapeutic strategy, particularly for cancer therapy.
UNASSIGNED: The recent advances of patented BRD4 degraders were summarized. The challenges, opportunities, and future directions for developing novel potent and selective BRD4 degraders are also discussed. The patents of BRD4 degraders were searched using the SciFinder and Cortellis Drug Discovery Intelligence database.
UNASSIGNED: BRD4 degraders exhibit superior efficacy and selectivity to BRD4 inhibitors, given their unique mechanism of protein degradation instead of protein inhibition. Excitingly, RNK05047 is now in phase I/II clinical trials, indicating that selective BRD4 protein degradation may offer a viable therapeutic strategy, particularly for cancer. Targeting BRD4 with small-molecule degraders provides a promising approach with the potential to overcome therapeutic resistance for treating various BRD4-associated diseases.

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with µ subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain \"vacuolar-trafficking zone,\" and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters.

Protein homeostasis depends on many fundamental processes including mRNA synthesis, translation, post-translational modifications, and proteolysis. In the late 70s and early 80s the discovery that the small 76 amino acid protein ubiquitin could be attached to target proteins via a multi-stage process involving ubiquitin-activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases, revealed an exciting new post-translational mechanism to regulate protein degradation. This cellular system was uncovered using biochemical methods by Avram Hershko, who would later won the Nobel prize for this discovery; however, the biological functions of ubiquitin ligases remained unknown for many years. It was initially described that ubiquitin modifies proteins at one or more lysine residues and once a long ubiquitin chain was assembled, proteins were degraded by the proteasome. Now we know that proteins can be mono-, multimono-, homotypic poly-, or heterotypic poly-ubiquitylated, each of which confers a specific signal that goes beyond protein degradation regulating additional key cellular functions such as signal transduction, protein localization, recognition of damaged proteins, etc.

The most frequently occurring protein modification in fish postmortem is oxidization, which further affects meat quality through multiple biochemical pathways. To investigate how hydroxyl radicals affect the structure of cathepsin H and its ability to break down myofibrillar proteins in Coregonus peled, cathepsin H was oxidized with 0, 0.1, 0.5, 1, 5, and 10 mM H2O2 and subsequently incubated with isolated myofibrillar proteins. The results showed that as the H2O2 concentration increased, the carbonyl and sulfhydryl contents of cathepsin H significantly increased and decreased, respectively. There were noticeable changes in the α-helix structures and a gradual reduction in UV absorbance and fluorescence intensity, indicating that oxidation can induce the cross-linking and aggregation of cathepsin H. These structural changes further reduced the activity of cathepsin H, reaching its lowest at 10 mM H2O2, which was 53.63% of the activity at 0 mM H2O2. Moreover, desmin and troponin-T all degraded at faster rates when cathepsin H and myofibrillar proteins were oxidized concurrently as opposed to when cathepsin H was oxidized alone. These findings provide vital insights into the interaction mechanism between oxidation, cathepsin H, as well as myofibrillar protein degradation, laying a groundwork for understanding the molecular mechanisms underlying changes in fish meat quality after slaughter and during processing.

This study explored the intrinsic relationship between the quality traits and protein-related property changes during the solar drying of shrimps (Penaeus vannamei). During drying, the shrimp exhibited a gradual decline in L*a*b* values and a notable increase in the hardness and chewiness. Proteins were degraded and oxidized. Especially, the trichloroacetic acid-soluble peptide and carbonyl contents increased, whereas the total sulfhydryl content decreased. The proportions of different types of intramolecular bonds were significantly changed. The ionic and hydrogen bonds greatly decreased to 10.72% and 9.05% and the hydrophobic and disulfide bonds notably increased to 19.38% and 28.19%, indicating the changes in the spatial structure of the protein and its denaturation during the drying process. The Pearson\'s correlation analysis showed that protein degradation and denaturation greatly affected the textural properties and protein oxidation caused color changes. The result of this work provides a theoretical support for improving the quality of shrimp products.

Proteome maintenance in contracting skeletal and cardiac muscles depends on the chaperone-regulating protein BAG3. Reduced BAG3 activity leads to muscle weakness and heart failure in animal models and patients. BAG3 and its chaperone partners recognize mechanically damaged muscle proteins and initiate their disposal through chaperone-assisted selective autophagy (CASA). However, molecular details of the force-dependent regulation of BAG3 have remained elusive so far. Here, we demonstrate that mechanical stress triggers the dephosphorylation of BAG3 in human muscle and in isolated cells. We identify force-regulated phospho-switches in BAG3 that control CASA complex assembly and CASA activity. Differential proteomics reveal RAB GTPases, which organize membrane traffic and fusion, as dephosphorylation-dependent interactors of BAG3. In fact, RAB7A and RAB11B are shown here to be essential for CASA in skeletal muscle cells. Moreover, BAG3 dephosphorylation is also observed upon induction of mitophagy, suggesting an involvement of the cochaperone in the RAB7A-dependent autophagic engulfment of damaged mitochondria in exercised muscle. Cooperation of BAG3 with RAB7A relies on a direct interaction of both proteins, which is regulated by the nucleotide state of the GTPase and by association with the autophagosome membrane protein LC3B. Finally, we provide evidence that BAG3 and RAB7A also cooperate in non-muscle cells and propose that overactivation of CASA in RAB7A-L129F patients contributes to the loss of peripheral neurons in Charcot-Marie-Tooth neuropathy.

Loss-of-function mutations in the C terminus of TPL2 kinase promote oncogenesis by impeding its proteasomal degradation, leading to sustained protein expression. However, the degradation mechanism for TPL2 has remained elusive. Through proximity-dependent biotin identification (BioID), we uncovered tripartite motif-containing 4 (TRIM4) as the E3 ligase that binds and degrades TPL2 by polyubiquitination of lysines 415 and 439. The naturally occurring TPL2 mutants R442H and E188K exhibit impaired TRIM4 binding, enhancing their stability. We further discovered that TRIM4 itself is stabilized by another E3 ligase, TRIM21, which in turn is regulated by KRAS. Mutant KRAS recruits RNF185 to degrade TRIM21 and subsequently TRIM4, thereby stabilizing TPL2. In the presence of mutant KRAS, TPL2 phosphorylates and degrades GSK3β, resulting in β-catenin stabilization and activation of the Wnt pathway. These findings elucidate the physiological mechanisms regulating TPL2 and its exploitation by mutant KRAS, underscoring the need to develop TPL2 inhibitors for KRAS-mutant cancers.