Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon on the outer chloroplast membrane) and the TOM (translocon on the outer mitochondrial membrane) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ adenosine triphosphatase CDC48, after which a previously uncharacterized cytosolic protein, transmembrane domain (TMD)-binding protein for tail-anchored outer membrane proteins (TTOP), binds to the exposed TMDs at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

Mesenchymal stem cells (MSCs) are the most widely used stem cells in regenerative medicine. They can be isolated from multiple sources, most commonly bone marrow and adipose tissue. MSCs derived from different sources show similar molecular and biological characteristics, but there is ongoing debate regarding the best source of MSCs and the potential biological differences between MSCs from different origins. Bone marrow derived-MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) share many molecular and immunomodulatory properties. In this study, we compared the levels of major immunomodulatory, adhesive, and migratory factors in human BM-MSCs and AD-MSCs under normal conditions, which will help determine the suitability and specificity of each type for certain therapeutic applications. WST1 assay and fluorescent assay SUC-LLVY-AMC were used to measure MSC proliferation and 26S proteasome activity, respectively. Western blotting, ELISA Assays, and bright field live imaging were also used. AD-MSCs and BM-MSCs exhibited similar morphology and proliferation rate. A significantly higher 26S proteasome activity was detected in AD-MSCs than in BM-MSCs. Levels of ICAM-1, integrin α5 and integrin α6 were significantly higher in AD-MSCs compared to BM-MSCs, while no significant difference in CXCR4 levels was observed. Expression of IDO and factor H was significantly higher in AD-MSCs, while CTLA-4 and IL-10 levels were higher in BM-MSCs. This indicates that AD-MSCs and BM-MSCs have different immunomodulatory and adhesion profiles. MSCs isolated from different sources may show differences in their biological and immunomodulatory properties, suggesting a potential suitability of certain MSCs type for specific conditions. Also, combination of different MSCs types could help optimize therapeutic outcomes.

Parkinson\'s disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.

The post-translational modification (PTM) of proteins by ubiquitination modulates many physiological processes in plants. As the major protein degradation pathway in plants, the ubiquitin-proteasome system (UPS) is considered a promising target for improving crop tolerance drought, high salinity, extreme temperatures, and other abiotic stressors. The UPS also participates in abiotic stress-related abscisic acid (ABA) signaling. E3 ligases are core components of the UPS-mediated modification process due to their substrate specificity. In this review, we focus on the abiotic stress-associated regulatory mechanisms and functions of different UPS components, emphasizing the participation of E3 ubiquitin ligases. We also summarize and discuss UPS-mediated modulation of ABA signaling. In particular, we focus our review on recent research into the UPS-mediated modulation of the abiotic stress response in major crop plants. We propose that altering the ubiquitination site of the substrate or the substrate-specificity of E3 ligase using genome editing technology such as CRISPR/Cas9 may improve the resistance of crop plants to adverse environmental conditions. Such a strategy will require continued research into the role of the UPS in mediating the abiotic stress response in plants.

Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system.
We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs.
The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the β5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased β5 (PSMB5) in MNs compared to HFFs.
The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit β5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation.

OBJECTIVE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has tragically claimed millions of lives through coronavirus disease 2019 (COVID-19), and there remains a critical gap in our understanding of the precise molecular mechanisms responsible for the associated fatality. One key viral factor of interest is the SARS-CoV-2 ORF3a protein, which has been identified as a potent inducer of host cellular proinflammatory responses capable of triggering the catastrophic cytokine storm, a primary contributor to COVID-19-related deaths. Moreover, ORF3a, much like the spike protein, exhibits a propensity for frequent mutations, with certain variants linked to the severity of COVID-19. Our previous research unveiled two distinct types of ORF3a mutant proteins, categorized by their subcellular localizations, setting the stage for a comparative investigation into the functional and mechanistic disparities between these two types of ORF3a variants. Given the clinical significance and functional implications of the natural ORF3a mutations, the findings of this study promise to provide invaluable insights into the potential roles undertaken by these mutant ORF3a proteins in the pathogenesis of COVID-19.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a \"loop surface\" with three protruding loops that form electrostatic interactions with ZnF, and a \"sheet surface\" featuring two β-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

OBJECTIVE: To evaluate the effects of resistance exercise training (RET) and/or glutamine supplementation (GS) on signaling protein synthesis in adult rat skeletal muscles.
METHODS: The following groups were studied: (1) control, no exercise (C); (2) exercise, hypertrophy resistance exercise training protocol (T); (3) no exercise, supplemented with glutamine (G); and (4) exercise and supplemented with glutamine (GT). The rats performed hypertrophic training, climbing a vertical ladder with a height of 1.1 m at an 80° incline relative to the horizontal with extra weights tied to their tails. The RET was performed three days a week for five weeks. Each training session consisted of six ladder climbs. The extra weight load was progressively increased for each animal during each training session. The G groups received daily L-glutamine by gavage (one g per kilogram of body weight per day) for five weeks. The C group received the same volume of water during the same period. The rats were euthanized, and the extensor digitorum longus (EDL) muscles from both hind limbs were removed and immediately weighed. Glutamine and glutamate concentrations were measured, and histological, signaling protein contents, and mRNA expression analyses were performed.
RESULTS: Supplementation with free L-glutamine increased the glutamine concentration in the EDL muscle in the C group. The glutamate concentration was augmented in the EDL muscles from T rats. The EDL muscle mass did not change, but a significant rise was reported in the cross-sectional area (CSA) of the fibers in the three experimental groups. The levels of the phosphorylated proteins (pAkt/Akt, pp70S6K/p70S6K, p4E-BP1/4E-BP1, and pS6/S6 ratios) were significantly increased in EDL muscles of G rats, and the activation of p4E-BP1 was present in T rats. The fiber CSAs of the EDL muscles in T, G, and GT rats were increased compared to the C group. These changes were accompanied by a reduction in the 26 proteasome activity of EDL muscles from T rats.
CONCLUSIONS: Five weeks of GS and/or RET induced muscle hypertrophy, as indicated by the increased CSAs of the EDL muscle fibers. The increase in CSA was mediated via the upregulated phosphorylation of Akt, 4E-BP1, p70S6k, and S6 in G animals and 4E-BP1 in T animals. In the EDL muscles from T animals, a decrease in proteasome activity, favoring a further increase in the CSA of the muscle fibers, was reported.

While 19S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14, forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma.