Ubiquitin-proteasome Proteolysis Research Articles

Deciphering the mechanism of E3 ubiquitin ligases in plant responses to abiotic and biotic stresses and perspectives on PROTACs for crop resistance.

With global climate change, it is essential to find strategies to make crops more resistant to different stresses and guarantee food security worldwide. E3 ubiquitin ligases are critical regulatory elements that are gaining importance due to their role in selecting proteins for degradation in the ubiquitin-proteasome proteolysis pathway. The role of E3 Ub ligases has been demonstrated in numerous cellular processes in plants responding to biotic and abiotic stresses. E3 Ub ligases are considered a class of proteins that are difficult to control by conventional inhibitors, as they lack a standard active site with pocket, and their biological activity is mainly due to protein-protein interactions with transient conformational changes. Proteolysis-targeted chimeras (PROTACs) are a new class of heterobifunctional molecules that have emerged in recent years as relevant alternatives for incurable human diseases like cancer because they can target recalcitrant proteins for destruction. PROTACs interact with the ubiquitin-proteasome system, principally the E3 Ub ligase in the cell, and facilitate proteasome turnover of the proteins of interest. PROTAC strategies harness the essential functions of E3 Ub ligases for proteasomal degradation of proteins involved in dysfunction. This review examines critical advances in E3 Ub ligase research in plant responses to biotic and abiotic stresses. It highlights how PROTACs can be applied to target proteins involved in plant stress response to mitigate pathogenic agents and environmental adversities.

A novel autism-associated UBLCP1 mutation impacts proteasome regulation/activity

The landscape of autism spectrum disorder (ASD) in Lebanon is unique because of high rates of consanguinity, shared ancestry, and increased remote consanguinity. ASD prevalence in Lebanon is 1 in 68 with a male-to-female ratio of 2:1. This study aims to investigate the impact of an inherited deletion in UBLCP1 (Ubiquitin-Like Domain-Containing CTD Phosphatase 1) on the ubiquitin-proteasome system (UPS) and proteolysis. Whole exome sequencing in a Lebanese family with ASD without pathogenic copy number variations (CNVs) uncovered a deletion in UBLCP1. Functional evaluation of the identified variant is described in fibroblasts from the affected. The deletion in UBLCP1 exon 10 (g.158,710,261CAAAG > C) generates a premature stop codon interrupting the phosphatase domain and is predicted as pathogenic. It is absent from databases of normal variation worldwide and in Lebanon. Wild-type UBLCP1 is widely expressed in mouse brains. The mutation results in decreased UBLCP1 protein expression in patient-derived fibroblasts from the autistic patient compared to controls. The truncated UBLCP1 protein results in increased proteasome activity decreased ubiquitinated protein levels, and downregulation in expression of other proteasome subunits in samples from the affected compared to controls. Inhibition of the proteasome by using MG132 in proband cells reverses alterations in gene expression due to the restoration of protein levels of the common transcription factor, NRF1. Finally, treatment with gentamicin, which promotes premature termination codon read-through, restores UBLCP1 expression and function. Discovery of an ASD-linked mutation in UBLCP1 leading to overactivation of cell proteolysis is reported. This, in turn, leads to dysregulation of proteasome subunit transcript levels as a compensatory response.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

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.

USP39 stabilizes β-catenin by deubiquitination and suppressing E3 ligase TRIM26 pre-mRNA maturation to promote HCC progression

Ubiquitin-specific protease 39(USP39) plays an important role in modulating pre-mRNA splicing and ubiquitin-proteasome dependent proteolysis as a member of conserved deubiquitylation family. Accumulating evidences prove that USP39 participates in the development of hepatocellular carcinoma (HCC). However, little is known about the mechanism especially deubiquitinating target of USP39 in regulating hepatocellular carcinoma (HCC) growth. Here, we prove that USP39 promotes HCC cell proliferation and migration by directly deubiquitin β-catenin, a key molecular of Wnt/β-catenin signaling pathway whose abnormal expression or activation results in several tumors, following its co-localization with USP39. In this process, the expression of E3 ligase TRIM26, which is proved to restrain HCC in our previous research, shows a decreasing trend. We further demonstrate that TRIM26 pre-mRNA splicing and maturation is inhibited by USP39, accompanied by its reduction of ubiquitinating β-catenin, facilitating HCC progression indirectly. In summary, our data reveal a novel mechanism in the progress of HCC that USP39 promotes the proliferation and migration of HCC through increasing β-catenin level via both direct deubiquitination and reducing TRIM26 pre-mRNA maturation and splicing, which may provide a new idea and target for clinical treatment of HCC.

Myelinosome organelles in pathological retinas: ubiquitous presence and dual role in ocular proteostasis maintenance.

The timely and efficient elimination of aberrant proteins and damaged organelles, formed in response to various genetic and environmental stressors, is a vital need for all cells of the body. Recent lines of evidence point out several non-classical strategies employed by ocular tissues to cope with aberrant constituents generated in the retina and in the retinal pigmented epithelium cells exposed to various stressors. Along with conventional strategies relying upon the intracellular degradation of aberrant constituents through ubiquitin-proteasome and/or lysosome-dependent autophagy proteolysis, two non-conventional mechanisms also contribute to proteostasis maintenance in ocular tissues. An exosome-mediated clearing and a myelinosome-driven secretion mechanism do not require intracellular degradation but provide the export of aberrant constituents and "waste proteins" outside of the cells. The current review is centered on the non-degradative myelinosome-driven secretion mechanism, which operates in the retina of transgenic Huntington's disease R6/1 model mice. Myelinosome-driven secretion is supported by rare organelles myelinosomes that are detected not only in degenerative Huntington's disease R6/1 retina but also in various pathological states of the retina and of the retinal pigmented epithelium. The intra-retinal traffic and inter-cellular exchange of myelinosomes was discussed in the context of a dual role of the myelinosome-driven secretion mechanism for proteostasis maintenance in different ocular compartments. Special focus was made on the interplay between degradative and non-degradative strategies in ocular pathophysiology, to delineate potential therapeutic approaches to counteract several vision diseases.

Atrogin-1 knockdown inhibits the autophagy-lysosome system in mammalian and avian myotubes

Atrogin-1 plays an important role in ubiquitin-proteasome proteolysis in vertebrate skeletal muscles. Recently, atrogin-1 has been shown to be involved in the autophagy-lysosome system, another proteolytic system, in the murine and fish hearts and skeletal muscles. With the aim to elucidate the effect of atrogin-1 on the autophagy-lysosome system in mammalian and avian skeletal muscles, this study has examined the effects of atrogin-1 knockdown on autophagy-lysosome-related proteins in C2C12 and chicken embryonic myotubes. Using the levels of microtubule-associated protein light chain 3 (LC3)-II protein, it was confirmed that atrogin-1 knockdown blocked the autophagic flux in both the myotubes. In addition, atrogin-1 knockdown in C2C12 myotubes significantly decreased the level of autophagy-related gene (ATG)12-ATG5 conjugate, which is supposedly necessary for the fusion of autophagosomes and lysosomes. Atrogin-1 knockdown also resulted in downregulation of forkhead box O3, a transcription factor for ATG12. These data suggest that atrogin-1 is essential for the normal autophagy-lysosome system in the striated muscles of vertebrates.

MERG1A Protein Abundance Increases in the Atrophied Skeletal Muscle of Denervated Mice, But Does Not Affect NFκB Activity.

Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.

The ERG1A Potassium Channel Induces Expression of Genes Related to Skeletal Muscle Atrophy in C2C12 Myotubes

The ERG1A potassium channel alternative splice variant is detected at low abundance in normal skeletal muscle; however, it is up-regulated in atrophying skeletal muscle, where it has been shown to modulate both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP). The pathways by which this modulation occurs are not known. Therefore, we transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). At 48 hours after viral treatments, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers. The cDNA libraries were sequenced on Illumina's NovaSeq platform and sequence quality was assessed using FastQC (v 0.11.7) for all samples. Quality trimming was performed with the FASTX-Toolkit (v 0.0.14) to remove bases with a Phred33 score of less than 30. The resulting reads of at least 50 bases were mapped against the reference genome using STAR. The mapping results and the annotation file for the reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1). The data demonstrate that HERG expression does produce numerous changes in the gene expression profile of C2C12 myotubes. Indeed, we find that HERG potentially modulates expression of numerous genes (see Table) connected with skeletal muscle atrophy, specifically ubiquitin proteasome proteolysis and with the cytokine interferon, which has been connected with muscle atrophy. The data suggest that HERG does play a role in modulation of protein degradation in skeletal muscle.

The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells

BackgroundSkeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells.MethodsWe transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student’s t test or two-way ANOVAs and SAS software as indicated.ResultsExpression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity.ConclusionsHuman ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

The ERG1a K+ Channel Increases Intracellular Calcium and Calpain Activity in C2C12 Myotubes

In skeletal muscle increased intracellular calcium concentration ([Ca2+]i; uM range) is necessary for excitation‐contraction coupling; however, smaller increases in [Ca2+]i (nM range) can modulate other physiological processes in non‐contracting muscle. Indeed, fluctuations in localized calcium concentration can serve as a second messenger. In contrast, inappropriate changes in [Ca2+]i can have detrimental effects on muscle tissue and are associated with numerous skeletal muscle pathologies, for example muscular dystrophy, amyotrophic lateral sclerosis, malignant hyperthermia, cancer cachexia, and atrophy. Therefore, [Ca2+]i must be tightly regulated in terms of time, space and amplitude for cellular processes to occur in a properly coordinated fashion. In earlier reports, we showed that the ERG1a K+ channel is upregulated in atrophying skeletal muscle and contributes to increased ubiquitin proteasome proteolysis. Here, using the ratiometric Ca2+ indicator fura‐2AM, we show that ERG1a expression in C2C12 myotubes produces an increase in basal [Ca2+]i as well as a transient increase in [Ca2+]i as a consequence of depolarization. We explored this transient increase in [Ca2+]i using pharmacological agents. The data demonstrate that the ERG1a‐induced increase is not sensitive to the L‐type calcium channel blocker nifedipine, suggesting that it does not result from modulation of Cav1.1 channels. To further support this data, immunoblots reveal that there is no change in Cav1.1 channel abundance. However, the data do demonstrate that the increase in [Ca2+]i is sensitive to the SERCA blocking agent thapsigargin, suggesting that the source of the calcium is the sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases basal calpain activity. In summary, to date the data show that ERG1a increases [Ca2+]i levels and suggest that this increase could occur, at least in part, as a result of release of calcium from sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases calpain activity, suggesting that the increase in intracellular calcium results in increased calpain activity and possibly contributes to the increased proteolysis that occurs in atrophic skeletal muscle.Support or Funding InformationU.S. Department of Defense, PRMRP Grant# PR170326This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Timed degradation of Mcl-1 controls mitotic cell death

ABSTRACTMitotic arrest can result in cell death through the process of apoptosis. We have shown by live-cell imaging that the ubiquitin-proteasome dependent proteolysis of the apoptotic regulator Mcl-1 under the control of the anaphase-promoting complex or cyclosome (APC/C) provides a timing mechanism that distinguishes prolonged mitotic arrest from normal mitosis.

Combination of Soy Protein, Amylopectin, and Chromium Stimulates Muscle Protein Synthesis by Regulation of Ubiquitin-Proteasome Proteolysis Pathway after Exercise.

The present study was undertaken to investigate the effect of the combination of soy protein, amylopectin, and chromium (SAC) on muscle protein synthesis and signal transduction pathways involved in protein synthesis (mTOR pathways, IGF-1, and AktSer473) and proteolysis (FOXO1Ser256; MURF1, MAFbx) after exercise. Thirty-five Wistar rats were randomly divided into five groups: (1) control (C); (2) exercise (E); (3) exercise + soy protein (3.1g/kg/day) (E + S); (4) exercise + soy protein + chromium (E + S + Cr); (5) exercise + soy protein + amylopectin + chromium (E + S + A + Cr). Post-exercise ingestion of SAC significantly increased the fractional rate of protein synthesis (FSR), insulin, glycogen, and amino acid levels with the highest effect observed in E + S + A + Cr group (P˂0.05). However, SAC supplementation decreased the lactic acid concentration (P˂0.05). A reduction in forkhead box protein O1 (FOXO1) and forkhead box protein O3 (FOXO3) (regulators of ubiquitin-related proteolysis) and muscle atrophy F-box (MAFbx) levels was noted after treatment with SAC (P< 0.05). Insulin-like growth factor 1(IGF-1) level was increased in the E + S, E + S + Cr, and E + S + A + Cr groups (P< 0.05). While the phosphorylation of 4E-BP1Thr37/46, AktSer473, mTORSer2448, and S6K1Thr389 levels increased after SAC supplementation, phosphorylated muscle ring finger 1 (MuRF-1, an E3-ubiquitin ligase gene) was found to be significantly lower compared with the E group (P˂0.05). These results indicate that SAC supplementation improves FSR, insulin, and glycogen levels after exercise. SAC improves protein synthesis by inhibiting the ubiquitin-proteasome pathway and inducing anabolic metabolism.

The ERG1 K+ Channel Protein Is More Abundant in the Denervated Skeletal Muscle of Rodents

Skeletal muscle atrophy is the loss of muscle mass and strength that occurs with normal aging, neural and muscle injuries, starvation and diseases such as diabetes, neuropathies, muscular dystrophy, cancer and sepsis cachexia, etc. The atrophy of skeletal muscle can be incapacitating and is related to increased disability, morbidity and mortality in people. We have shown that the ERG1a alternative splice variant of the ERG1 K+ channel is up‐regulated in the skeletal muscle of mice experiencing atrophy as a consequence of cancer cachexia and disuse (i.e., hind limb unweighting, a condition in which muscle action potentials are reduced). In fact, the ERG1 protein is up‐regulated after 4 days of hind limb unweighting, which is prior to a significant loss in muscle cross sectional area, strongly suggesting ERG1 is involved in the onset of muscle loss. Further, we have shown that the ERG1a channel contributes to protein breakdown in muscle by increasing both ubiquitin proteasome proteolysis (UPP) and intracellular calcium concentration with a subsequent increase in calpain activity. Because skeletal muscle atrophy occurs rapidly in denervated skeletal muscle, we hypothesized that ERG1 would also increase in denervated muscles. To test this hypothesis, we performed sciatic nerve transection on mice and rats and collected gastrocnemius and soleus muscles at 0, 4, 7 and 14 days after surgery. Decreased muscle weight and myofiber cross sectional area demonstrate that the muscles were successfully denervated and underwent atrophy. Immunoblot with anti‐ERG1 antibodies demonstrates that indeed the ERG1 protein increases post denervation in mice. Further, immunohistochemistry demonstrates that ERG1 protein also increases in denervated soleus of rats. Interestingly, confocal microscopy analysis of these tissues shows that the ERG1 protein is localized in the sarcolemma and t‐tubules of denervated rat skeletal muscle as it is in heart. The t‐tubule location places it in the vicinity of the calcium release apparatus where it can affect calcium release. We conclude that ERG1 protein is upregulated in the t‐tubules and sarcolemma of denervated muscle and, because it is up‐regulated in other atrophy models, ERG1 is a consistent marker of skeletal muscle atrophy.Support or Funding InformationThis research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy.

Progranulin Controls Sepsis via C/EBPα-Regulated Il10 Transcription and Ubiquitin Ligase/Proteasome-Mediated Protein Degradation.

Progranulin (PGRN) is a widely expressed, pleiotropic protein that is involved in diverse biological processes, including cellular proliferation, neuron development, and wound healing. However, the role of PGRN in the regulation of pathogen-induced systemic inflammation and the mechanisms involved have not been established. In this study, we show that PGRN-deficient mice display heightened mortality in models of polymicrobial sepsis and endotoxinemia, with increased tissue levels of inflammatory cytokines and reduced IL-10 production. Conversely, administration of rPGRN decreases the susceptibility of PGRN-deficient mice to LPS-induced endotoxemic shock and augments IL-10 production by LPS-activated macrophages in a TNFR-dependent manner. Molecular analysis reveals a direct role of the transcription factor C/EBPα in PGRN-regulated IL-10 expression. C/EBPα-deficient macrophages produce less IL-10 in response to LPS. Furthermore, mice deficient in C/EBPα in hematopoietic cells are highly vulnerable to LPS-induced septic shock. Lastly, the defective IL-10 production by PGRN-deficient cells is primarily due to reduced C/EBPα protein stability via the E3 ubiquitin-conjugating enzyme E6AP and proteasome-mediated degradation. To our knowledge, this study provides the first evidence that PGRN is a nonredundant regulator of systemic inflammation via modulating the levels and activity of C/EBPα, IL-10, and the ubiquitin-proteasome proteolysis pathway. The results bear strong and profound implications for PGRN insufficiency and its mutation-associated systemic and organ-specific inflammatory human diseases.

Alanyl-glutamine supplementation regulates mTOR and ubiquitin proteasome proteolysis signaling pathways in piglets

ObjectiveThe aim of the present study was to investigate the effects of the alanyl-glutamine dipeptide (Ala-Gln) or the combination supplementation of free alanine and glutamine (Ala+Gln) on the mammalian target of rapamycin (mTOR) and ubiquitin-proteasome proteolysis (UPP) signaling pathways in piglets. MethodsWe randomly allocated 180 piglets to three treatments with three replicates of 20 piglets each, fed with diets containing 0.62% Ala, 0.5% Ala-Gln, 0.21% Ala+0.34% Gln, respectively. The duration of the experiment was 28 d. ResultsThe results showed that Ala-Gln increased average daily gain of piglets, and decreased the ratio of feed to gain (P < 0.05). Ala-Gln supplementation increased the concentrations of Gln and glutamate and decreased the activity of glutamine synthetase in liver and skeletal muscle (P < 0.05). Ala-Gln increased the expression of glutaminase and glutamate dehydrogenate (P < 0.05). The increased phosphorylation of eIF-4 E binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1) in Ala-Gln treatment were associated with phosphorylation of the mTOR in liver and skeletal muscle. Ala+Gln did not affect the phosphorylation abundances of mTOR, 4E-BP1, or S6K1 (P > 0.05). Ala-Gln supplementation inhibited the mRNA expressions of MAFbx and MuRF1 in skeletal muscle of piglets (P < 0.05). ConclusionTaken together, Ala-Gln supplementation improved the growth performance of piglets, enhanced the metabolism of Gln, upregulated protein synthetic signaling in liver and skeletal muscle and decreased protein degradative signaling in muscle of piglets. Moreover, these effects of Ala-Gln were more effective than those of Ala+Gln.

TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation.

Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice. This finding shows the functional significance of TSGΔ154-1054 in preventing the ubiquitin-proteasome proteolysis of TSG101, which increases tumor malignancy and hints at its potential as a therapeutic target in cancer treatment.

Abstract LB-026: Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1

Abstract Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein in cooperation with numerous tyrosine kinases to regulate various cellular signaling and functions. Although the regulatory mechanisms on the activation of Grb7 have been documented, the molecular mechanism governing Grb7 stability and its functional consequence is still not clearly understood. Here, we observed a novel negative regulatory mechanism of Grb7 by peptidyl-prolyl cis/trans isomerases Pin1 at the post-translational level. The Grb7 phosphorylation on the Ser194-Pro motif facilitated its binding to the WW domain of Pin1 and subsequently ubiquitination on Grb7, which, in turn, enhanced the process of proteosome-dependent proteolysis. Moreover, the interaction between Grb7 and Pin1 is dependent on the phosphorylation that is mediated by c-Jun N-terminal kinases MAPK. Both the phosphorylated Ser/Thr-Pro motif binding module and the peptidyl-prolyl cis/trans isomerase activity of Pin1 is essentail for Pin1-mediated Grb7 ubiquitin-proteasome proteolysis. By contrast, inhibition of Pin by lentiviral-mediated gene silencing resulted in accumulation of Grb7 protein as well as prolonged Grb7 protein stability. A stable Grb7S194A mutant that cannot be bound to and modulated by Pin1 utilize its potential to influence cell cycle progression. Our finding revealed that the Pin1/Grb7 complex formation enables negatively regulating Grb7-mediated cell proliferation due to the influence of Grb7 protein stability. Note: This abstract was not presented at the meeting. Citation Format: Yu-Ling Tai, Tang-Long Shen. Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-026. doi:10.1158/1538-7445.AM2015-LB-026

The Sick and the Weak: Neuropathies/Myopathies in the Critically Ill.

Critical illness polyneuropathies (CIP) and myopathies (CIM) are common complications of critical illness. Several weakness syndromes are summarized under the term intensive care unit-acquired weakness (ICUAW). We propose a classification of different ICUAW forms (CIM, CIP, sepsis-induced, steroid-denervation myopathy) and pathophysiological mechanisms from clinical and animal model data. Triggers include sepsis, mechanical ventilation, muscle unloading, steroid treatment, or denervation. Some ICUAW forms require stringent diagnostic features; CIM is marked by membrane hypoexcitability, severe atrophy, preferential myosin loss, ultrastructural alterations, and inadequate autophagy activation while myopathies in pure sepsis do not reproduce marked myosin loss. Reduced membrane excitability results from depolarization and ion channel dysfunction. Mitochondrial dysfunction contributes to energy-dependent processes. Ubiquitin proteasome and calpain activation trigger muscle proteolysis and atrophy while protein synthesis is impaired. Myosin loss is more pronounced than actin loss in CIM. Protein quality control is altered by inadequate autophagy. Ca(2+) dysregulation is present through altered Ca(2+) homeostasis. We highlight clinical hallmarks, trigger factors, and potential mechanisms from human studies and animal models that allow separation of risk factors that may trigger distinct mechanisms contributing to weakness. During critical illness, altered inflammatory (cytokines) and metabolic pathways deteriorate muscle function. ICUAW prevention/treatment is limited, e.g., tight glycemic control, delaying nutrition, and early mobilization. Future challenges include identification of primary/secondary events during the time course of critical illness, the interplay between membrane excitability, bioenergetic failure and differential proteolysis, and finding new therapeutic targets by help of tailored animal models.

Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.