Regulation Of Protein Turnover Research Articles

Differential Induction Of Regulators Of Protein Turnover During C26-induced Cancer Cachexia Between Biological Sexes

Differential Induction Of Regulators Of Protein Turnover During C26-induced Cancer Cachexia Between Biological Sexes

Integrating multi-omics data reveals function and therapeutic potential of deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).

L-Arginine/nitric oxide regulates skeletal muscle development via muscle fibre-specific nitric oxide/mTOR pathway in chickens.

L-Arginine (L-Arg), the precursor of nitric oxide (NO), plays an important role in muscle function. Fast-twitch glycolytic fibres are more susceptible to age-related atrophy than slow-twitch oxidative fibres. The effect of L-Arg/NO on protein metabolism of fast- and slow-twitch muscle fibres was evaluated in chickens. In Exp. 1, 48 chicks at 1 day old were divided into 4 groups of 12 birds and subjected to 4 treatments: basal diet without supplementation or supplemented with 1% L-Arg, and water supplemented with or without L-nitro-arginine methyl ester (L-NAME, 18.5 mM). In Exp. 2, 48 chicks were divided into 4 groups of 12 birds fed with the basal diet and subjected to the following treatments: tap water (control), tap water supplemented with L-NAME (18.5 mM), or molsidomine (MS, 0.1 mM), or 18.5 mM L-NAME + 0.1 mM MS (NAMS). The regulatory effect of L-Arg/NO was further investigated in vitro with myoblasts obtained from chicken embryo pectoralis major (PM) and biceps femoris (BF). In vivo, dietary L-Arg supplementation increased breast (+14.94%, P < 0.05) and thigh muscle mass (+23.40%, P < 0.05); whereas, MS treatment had no detectable influence. However, L-NAME treatment blocked the beneficial influence of L-Arg on muscle development. L-Arg decreased (P < 0.05) protein synthesis rate, phosphorylated mTOR and ribosomal protein S6 kinase beta-1 (p70S6K) levels in breast muscle, which was recovered by L-NAME treatment. In vitro, L-Arg or sodium nitroprusside (SNP) reduced protein synthesis rate, suppressed phosphorylated mTOR/p70S6K and decreased atrogin-1 and muscle RING finger 1 (MuRF1) in myoblasts from PM muscle (P < 0.05). L-NAME abolished the inhibitory effect of L-Arg on protein synthesis and the mTOR/p70S6K pathway. However, myoblasts from BF muscle showed the weak influence. Moreover, blocking the mTOR/p70S6K pathway with rapamycin suppressed protein synthesis of the 2 types of myoblasts; whereas, the protein expression of atrogin-1 and MuRF1 levels were restricted only in myoblasts from PM muscle. In conclusion, L-Arg/NO/mTOR/p70S6K pathway enhances protein accumulation and muscle development in fast-twitch glycolytic muscle in chickens. L-Arg/NO regulates protein turnover in a muscle fibre specific way, which highlights the potential clinical application in fast-twitch glycolytic muscle fibres.

Essential Amino Acid Regulation of Protein Turnover to Support Skeletal Muscle Health with Age

Skeletal muscle (SM) is vital for both long term health and quality of life. Recent research suggests an increase in catabolic signals with age triggers pathologic conditions, such as sarcopenia. Although results from in vitro studies model how essential amino acids (EAA) can regulate muscle protein synthesis (MPS), the relevance of these models to muscle protein breakdown (MPB), and the presence of physiological plasma amino acid concentrations (PAA) remain to be established. Therefore, the objective of this study was to determine the effects of EAA with (0.2, 1.0, and 3.0 x PAA) in a young (passages 2‐10) and aging (passages 16‐24) C2C12 murine muscle cell model. We hypothesized that increased levels of EAA will increase MPS in aging cells and suppress MPB via the mTORC1 pathway when compared to young cells. Myoblasts were seeded (1x105) into 6‐well plates and differentiated into myotubes when they reached 80% confluency. Myotubes were serum and AA starved for 24 hours before receiving one of the following treatments: control (CON), 0.2 x PAA, 1.0 x PAA, 3.0 x PAA with or without rapamycin (100 nm; RAP) for 1, 6, and 24 hours. All treatments were performed in triplicate and then each experiment was repeated three times, yielding nine wells per treatment. Proteins related to sensing of AA and protein synthesis and degradation were assessed using Western Blot. Rapamycin inhibited phosphorylation of p70 S6 kinase 1 (p70S6K1) and 4E binding protein 1 (4E‐BP1), downstream targets of mTORC1, in both CON and AA‐treated young and aged cells. Phosphorylation of 4EBP1 and p70S6K1 was significantly increased (p&lt;0.05) in both young and aged cells following all PAA treatments. However, there was no significance after 24 hours of AA supplementation in aged cells. Particularly, after 6 hours of treatment all doses significantly increased (p&lt;0.0001) phosphorylation of p70S6K1 in both young and aged cells. 0.2 x PAA and 3.0 x PAA elicited a significantly greater response in 4EBP1 phosphorylation in young cells compared to the CON after 6 hours of treatment, whereas a dose of 3.0 x PAA induced a similar response in aged cells. In conclusion, in a C2C12 myotube model, increasing EAA concentrations downregulates the expression of translation initiation factors in young and aging cells. Specifically, it takes a 3.0 x PAA to elicit a response in aging cells, whereas young cells respond to lower physiological concentrations of 0.2 x PAA and 1.0 x PAA. Therefore, it can be proposed increasing EAA may be effective for adjusting the rate of MPS and MPB via the mTORC1 pathway in both young and aging muscle cells.

Altered Mitochondrial Protein Homeostasis and Proteinopathies.

Increasing evidence implicates mitochondrial dysfunction as key in the development and progression of various forms of neurodegeneration. The multitude of functions carried out by mitochondria necessitates a tight regulation of protein import, dynamics, and turnover; this regulation is achieved via several, often overlapping pathways that function at different levels. The development of several major neurodegenerative diseases is associated with dysregulation of these pathways, and growing evidence suggests direct interactions between some pathogenic proteins and mitochondria. When these pathways are compromised, so is mitochondrial function, and the resulting deficits in bioenergetics, trafficking, and mitophagy can exacerbate pathogenic processes. In this review, we provide an overview of the regulatory mechanisms employed by mitochondria to maintain protein homeostasis and discuss the failure of these mechanisms in the context of several major proteinopathies.

Molecular Regulators of Muscle Mass and Mitochondrial Remodeling Are Not Influenced by Testosterone Administration in Young Women.

Testosterone (T) administration has previously been shown to improve muscle size and oxidative capacity. However, the molecular mechanisms underlying these adaptations in human skeletal muscle remain to be determined. Here, we examined the effect of moderate-dose T administration on molecular regulators of muscle protein turnover and mitochondrial remodeling in muscle samples collected from young women. Forty-eight healthy, physically active, young women (28 ± 4 years) were assigned in a random double-blind fashion to receive either T (10 mg/day) or placebo for 10-weeks. Muscle biopsies collected before and after the intervention period were divided into sub-cellular fractions and total protein levels of molecular regulators of muscle protein turnover and mitochondrial remodeling were analyzed using Western blotting. T administration had no effect on androgen receptor or 5α-reductase levels, nor on proteins involved in the mTORC1-signaling pathway (mTOR, S6K1, eEF2 and RPS6). Neither did it affect the abundance of proteins associated with proteasomal protein degradation (MAFbx, MuRF-1 and UBR5) and autophagy-lysosomal degradation (AMPK, ULK1 and p62). T administration also had no effect on proteins in the mitochondria enriched fraction regulating mitophagy (Beclin, BNIP3, LC3B-I, LC3B-II and LC3B-II/I ratio) and morphology (Mitofilin), and it did not alter the expression of mitochondrial fission- (FIS1 and DRP1) or fusion factors (OPA1 and MFN2). In summary, these data indicate that improvements in muscle size and oxidative capacity in young women in response to moderate-dose T administration cannot be explained by alterations in total expression of molecular factors known to regulate muscle protein turnover or mitochondrial remodeling.

Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis

Proteins expressed by the paired box gene 9 (PAX9) and Msh Homeobox 1 (MSX1) are intimately involved in tooth development (odontogenesis). The regulation of PAX9 and MSX1 protein turnover by deubiquitinating enzymes (DUBs) plausibly maintain the required levels of PAX9 and MSX1 during odontogenesis. Herein, we used a loss-of-function CRISPR-Cas9-mediated DUB KO library kit to screen for DUBs that regulate PAX9 and MSX1 protein levels. We identify and demonstrate that USP49 interacts with and deubiquitinates PAX9 and MSX1, thereby extending their protein half-lives. On the other hand, the loss of USP49 reduces the levels of PAX9 and MSX1 proteins, which causes transient retardation of odontogenic differentiation in human dental pulp stem cells and delays the differentiation of human pluripotent stem cells into the neural crest cell lineage. USP49 depletion produced several morphological defects during tooth development, such as reduced dentin growth with shrunken enamel space, and abnormal enamel formation including irregular mineralization. In sum, our results suggest that deubiquitination of PAX9 and MSX1 by USP49 stabilizes their protein levels to facilitate successful odontogenesis.

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation.

BackgroundMost patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. The mechanisms underlying muscle loss in cancer cachexia remain elusive. Pancreatic tumour organoids are 3D cell culture models that retain key characteristics of the parent tumour. We aimed to investigate the effect of pancreatic tumour organoid‐derived factors on processes that determine skeletal muscle mass, including the regulation of muscle protein turnover and myogenesis.MethodsConditioned medium (CM) was collected from human pancreatic cancer cell lines (PK‐45H, PANC‐1, PK‐1, and KLM‐1), pancreatic tumour organoid cultures from a severely cachectic (PANCO‐9a) and a non‐cachectic patient (PANCO‐12a), and a normal pancreas organoid culture. Differentiating C2C12 myoblasts and mature C2C12 myotubes were exposed to CM for 24 h or maintained in control medium. In myotubes, NF‐kB activation was monitored using a NF‐κB luciferase reporter construct, and mRNA expression of E3‐ubiquitin ligases and REDD1 was analysed by RT‐qPCR. C2C12 myoblast proliferation and differentiation were monitored by live cell imaging and myogenic markers and myosin heavy chain (MyHC) isoforms were assessed by RT‐qPCR.ResultsWhereas CM from PK‐1 and KLM‐1 cells significantly induced NF‐κB activation in C2C12 myotubes (PK‐1: 3.1‐fold, P < 0.001; KLM‐1: 2.1‐fold, P = 0.01), Atrogin‐1/MAFbx and MuRF1 mRNA were only minimally and inconsistently upregulated by the CM of pancreatic cancer cell lines. Similarly, E3‐ubiquitin ligases and REDD1 mRNA expression in myotubes were not altered by exposure to pancreatic tumour organoid CM. Compared with the control condition, CM from both PANCO‐9a and PANCO‐12a tumour organoids increased proliferation of myoblasts, which was accompanied by significant downregulation of the satellite cell marker paired‐box 7 (PAX7) (PANCO‐9a: −2.1‐fold, P < 0.001; PANCO‐12a: −2.0‐fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO‐9a: −2.1‐fold, P < 0.001; PANCO‐12a: −1.8‐fold, P < 0.001) after 48 h of differentiation. Live cell imaging revealed accelerated alignment and fusion of myoblasts exposed to CM from PANCO‐9a and PANCO‐12a, which was in line with significantly increased Myomaker mRNA expression levels (PANCO‐9a: 2.4‐fold, P = 0.001; PANCO‐12a: 2.2‐fold, P = 0.004). These morphological and transcriptional alterations were accompanied by increased expression of muscle differentiation markers such as MyHC‐IIB (PANCO‐9a: 2.5‐fold, P = 0.04; PANCO‐12a: 3.1‐fold, P = 0.006). Although the impact of organoid CM on myogenesis was not associated with the cachexia phenotype of the donor patients, it was specific for tumour organoids, as CM of control pancreas organoids did not modulate myogenic fusion.ConclusionsThese data show that pancreatic tumour organoid‐derived factors alter the kinetics of myogenesis, which may eventually contribute to impaired muscle mass maintenance in cancer cachexia.

Multi-targeted therapy of cancer by omega-3 fatty acids-an update

Low in dietary ω3 polyunsaturated fatty acid (PUFA) consumption has been associated with increased incidence of cancers. Many basic and clinical studies have been conducted over the last several decades. We previously reviewed multi-targeted therapy of cancer by omega-3 fatty acids in 2008, and since hundreds of new clinical trials are being conducted to validate the effectiveness of ω3 PUFA in cancer therapy. Because of the availability of such large amount of clinical trial data, in this update we summarize clinical data, sort out trials that show promising results, and discuss potential mechanism(s) responsible for the clinical outcomes. It appears that ω3 PUFA mainly affects cancer-associated symptoms, namely cachexia, inflammation, neuropathy, post operative complications and quality of life. Mechanisms responsible for these effects are possible regulation of skeletal muscle protein turnover, inflammatory response and neuron cell survive by ω3 PUFA.

Lower IGFBP-1 Plasma Levels in Mothers of Autistic Children

IGF-I is an important growth factor and hormone which regulates protein turnover as well as having potent mitogenic and differentiating effects on most cell types.IGF binding proteins (IGFBP) act as carrier proteins and regulators by controlling bioavailability of IGF-I to tissues. We used immune-arrays to measure IGFBP-1in mothers of autistic children and found that the levels were significantly lower than levels in mothers of non-autistic children. IGF has been found to be decreased in autism. We suggest that these low levels are associated with low maternal IGFBP-1.

Microscopic Imaging of Endosomal Trafficking of ABA Receptors.

The abscisic acid (ABA) is a key hormone for stress tolerance. The balance between growth/development and stress responses is crucial for the optimal course of plant life meaning that plants need to control the timing and extent of ABA pathway activation. In this regard, protein turnover regulation by means of both the ubiquitin-proteasome system (UPS) and non-26S proteasome endomembrane trafficking pathways, plays a critical role in the regulation of ABA signaling activation and deactivation. Over the last few years, the ubiquitination of ABA receptors PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) at the plasma membrane by the RING between RING fingers (RBR)-type E3 ligase RING FINGER OF SEED LONGEVITY1 (RSL1) triggering their internalization through the clathrin-mediated endocytosis (CME) pathway, followed by their endosomal trafficking and delivery to the vacuole for degradation, was reported. For this process, the direct role of some components of the endosomal sorting complex required for transport (ESCRT) machinery, that is, FYVE DOMAIN-CONTAINING PROTEIN 1 (FYVE1)/FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) and VACUOLAR PROTEIN SORTING23A (VPS23A) members of ESCRT-I complex, and ALG-2 INTERACTING PROTEIN-X (ALIX) associated protein of ESCRT-III, was reported. In this chapter, we will detail two methods for imaging endosomal trafficking of ABA receptor proteins by confocal microscopy: (a) colocalization of GFP-PYL4 (also known as RCAR10) and CLATHRIN LIGHT CHAIN 2 (CLC2)-mOrange in clathrin-coated vesicles in Nicotiana benthamiana leaf cells and (b) localization of GFP-PYL4 into Wortmannin (WM)-enlarged late endosomes in Arabidopsis thaliana root cells.

Inhibitory Effects of Chrysanthemum (Chrysanthemum morifolium Ramat.) Extract and Its Active Compound Isochlorogenic Acid A on Sarcopenia.

Sarcopenia, age-related muscle atrophy, weakening muscle strength, and exercise capacity, generally accompany imbalances in protein metabolism. Chrysanthemum morifolium Ramat. extract (CME) and its active compound, isochlo-rogenic acid A (IcA), have been reported to have anti-oxidative, anti-diabetic, and neuroprotective effects. However, the roles of CME and IcA in the regulation of muscle protein turnover-related signaling pathways to attenuate sarcopenia have not been explored. In this study, we investigated CME and IcA based regulation of protein turnover in synthesizing muscle in vitro and in vivo. At the molecular level, CME and IcA promoted phosphorylation of PI3K/Akt and mTOR pathways, which stimulate synthesis of muscle proteins, and suppressed FoxO3a and E3 ubiquitin ligases during protein degrada-tion. In vivo, CME and IcA increased grip strength, exercise capacity, muscle mass and volume, and cross-sectional area of myofibers in middle-aged C57BL/6J mice. These results suggest that CME and IcA may have roles as functional food supplements for delaying sarcopenia by enhancing muscle mass recovery and function.

Oyster Hydrolysates Attenuate Muscle Atrophy via Regulating Protein Turnover and Mitochondria Biogenesis in C2C12 Cell and Immobilized Mice.

Sarcopenia, also known as skeletal muscle atrophy, is characterized by significant loss of muscle mass and strength. Oyster (Crassostrea gigas) hydrolysates have anti-cancer, antioxidant, and anti-inflammation properties. However, the anti-sarcopenic effect of oyster hydrolysates remains uninvestigated. Therefore, we prepared two different oyster hydrolysates, namely TGPN and PNY. This study aimed to determine the anti-muscle atrophy efficacy and molecular mechanisms of TGPN and PNY on both C2C12 cell lines and mice. In vitro, the TGPN and PNY recovered the dexamethasone-induced reduction in the myotube diameters. In vivo, TGPN and PNY administration not only improved grip strength and exercise endurance, but also attenuated the loss of muscle mass and muscle fiber cross-sectional area. Mechanistically, TGPN and PNY increased the expression of protein synthesis-related protein levels via phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin pathway, and reduced the expression of protein degradation-related protein levels via the PI3K/Akt/forkhead box O pathway. Also, TGPN and PNY stimulated NAD-dependent deacetylase sirtuin-1(SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1,2, mitochondrial transcription factor A, along with mitochondrial DNA content via SIRT1/PGC-1α signaling. These findings suggest oyster hydrolysates could be used as a valuable natural material that inhibits skeletal muscle atrophy via regulating protein turnover and mitochondrial biogenesis.

Design, synthesis and biological evaluation of 7-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-2,3-dihydro-1H-inden-1-one derivatives as potent FAK inhibitors for the treatment of ovarian cancer

Focal adhesion kinase (FAK) promotes tumor progression by intracellular signal transduction and regulation of gene expression and protein turnover, which is a compelling therapeutic target for various cancer types, including ovarian cancer. However, the clinical responses of FAK inhibitors remain unsatisfactory. Here, we describe the discovery of FAK inhibitors using a scaffold hopping strategy. Structure–activity relationship (SAR) exploration identified 36 as a potent FAK inhibitor, which exhibited inhibitory activities against FAK signaling in vitro. Treatment with 36 not only decreased migration and invasion of PA-1 cells, but also reduced expression of MMP-2 and MMP-9. Moreover, 36 inhibited tumor growth and metastasis, and no obvious adverse effects were observed during the in vivo study. These results revealed the potential of FAK inhibitor 36 for treatment of ovarian cancer.

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

ITRAQ-based proteomic analysis provides insights into the molecular mechanisms of rice formyl tetrahydrofolate deformylase in salt response.

A new molecular mechanism of tetrahydrofolate deformylase involved in the salt response presumably affects mitochondrial and chloroplast function by regulating energy metabolism and accumulation of reactive oxygen species. High salinity severely restrains plant growth and development, consequently leading to a reduction in grain yield. It is therefore critical to identify the components involved in plant salt resistance. In our previous study, we identified a rice leaf early-senescence mutant hpa1, which encodes a formyl tetrahydrofolate deformylase (Xiong et al. in Sci China Life Sci 64(5):720-738, 2021). Here, we report that HPA1 also plays a role in the salt response. To explore the molecular mechanism of HPA1 in salt resistance, we attempted to identify the differentially expressed proteins between wild type and hpa1 mutant for salinity treatment using an iTRAQ-based comparative protein quantification approach. A total of 4598 proteins were identified, of which 279 were significantly altered, including 177 up- and 102 down-regulated proteins. A functional analysis suggested that the 279 differentially expressed proteins are involved mainly in the regulation of oxidative phosphorylation, phenylpropanoid biosynthesis, photosynthesis, posttranslational modifications, protein turnover and energy metabolism. Moreover, a deficiency in HPA1 impaired chlorophyll metabolism and photosynthesis in chloroplasts and affected the electron flow of the electron transport chain in mitochondria. These changes led to abnormal energy metabolism and accumulation of reactive oxygen species, which may affect the permeability and integrity of cell membranes, leading to cell death. In addition, the results were verified by transcriptional or physiological experiments. Our results provide an insight into a new molecular mechanism of the tetrahydrofolate cycle protein formyl tetrahydrofolate deformylase, which is involved in the salt response, presumably by affecting mitochondrial and chloroplast function regulating energy metabolism and accumulation of reactive oxygen species under salt stress.

Protease activity is maintained in Nepenthes ampullaria digestive fluids depleted of endogenous proteins with compositional changes.

Nepenthes ampullaria is a unique carnivorous tropical pitcher plant with the detritivorous capability of sequestering nutrients from leaf litter apart from being insectivorous. The changes in the protein composition and protease activity of its pitcher fluids during the early opening of pitchers (D0 and D3C) were investigated via a proteomics approach and a controlled protein depletion experiment (D3L). A total of 193 proteins were identified. Common proteins such as pathogenesis-related protein, proteases (Nep [EC:3.4.23.12], SCP [EC:3.4.16.-]), peroxidase [EC:1.11.1.7], GDSL esterase/lipase [EC:3.1.1.-], and purple acid phosphatase [EC:3.1.3.2] were found in high abundance in the D0 pitchers and were replenished in D3L samples. This reflects their importance for biological processes upon pitcher opening. Meanwhile, prey-inducible chitinases [EC:3.2.1.14] were found in D0 but not in D3C and D3L samples, which suggests their degradation in the absence of prey. Protease activity assays demonstrated the replenishment of proteases in D3L with similar levels of proteolytic activities to that of D3C samples. This supports a feedback mechanism and signaling in the molecular regulation of endogenous protein secretion, turnover, and activity in Nepenthes pitcher fluids. Furthermore, we also discovered several new enzymes (XTH [EC:2.4.1.207], PAE [EC:3.1.1.98]) with possible functions in cell wall degradation that could contribute to the detritivory habit of N. ampullaria.

Pharmacological inhibition of BAG3-HSP70 with the proposed cancer therapeutic JG-98 is toxic for cardiomyocytes.

The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, whichinhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

Ubiquitin-Specific Proteases: Players in Cancer Cellular Processes.

Ubiquitination represents a post-translational modification (PTM) essential for the maintenance of cellular homeostasis. Ubiquitination is involved in the regulation of protein function, localization and turnover through the attachment of a ubiquitin molecule(s) to a target protein. Ubiquitination can be reversed through the action of deubiquitinating enzymes (DUBs). The DUB enzymes have the ability to remove the mono- or poly-ubiquitination signals and are involved in the maturation, recycling, editing and rearrangement of ubiquitin(s). Ubiquitin-specific proteases (USPs) are the biggest family of DUBs, responsible for numerous cellular functions through interactions with different cellular targets. Over the past few years, several studies have focused on the role of USPs in carcinogenesis, which has led to an increasing development of therapies based on USP inhibitors. In this review, we intend to describe different cellular functions, such as the cell cycle, DNA damage repair, chromatin remodeling and several signaling pathways, in which USPs are involved in the development or progression of cancer. In addition, we describe existing therapies that target the inhibition of USPs.

Expression of specific signaling components related to muscle protein turnover and of branched-chain amino acid catabolic enzymes in muscle and adipose tissue of preterm and term calves

Postnatal metabolism depends on maturation of key metabolic pathways around birth. In this regard, endogenous glucose production is impaired in calves born preterm. Concerning protein metabolism, the rates of protein turnover are greater during the neonatal period than at any other period of postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system (UPS) are considered as the major regulators of cellular protein turnover. The objectives of this study were to investigate (1) the changes in plasma AA profiles, (2) the mRNA abundance of mTOR signaling and UPS-related genes in skeletal muscle, and (3) the mRNA abundance of branched-chain AA (BCAA) catabolic enzymes in skeletal muscle and adipose tissue in neonatal calves with different degree of maturation during the transition to extrauterine life. Calves (n = 7/treatment) were born either preterm (PT; delivered by cesarean section 9 d before term) or at term (T; spontaneous vaginal delivery) and were left unfed for 1 d. Calves in treatment TC were also spontaneously born but were fed colostrum and transition milk for 4 d. Blood samples were collected from all calves at birth and at 24 h of life. Additional blood samples were taken 2 h after feeding (26 h of life) for PT and T calves, and on d 4 of life for TC, to determine plasma glucose, urea, and AA. Tissue samples from 3 muscles [M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM)], and kidney fat were collected following euthanasia at 26 h after birth (PT, T) or on d 4 of life (TC) at 2 h after feeding. The concentrations of the majority of plasma AA (Ala, Gln, Asn, Cit, Lys, Orn, Thr, and Tyr), nonessential AA, and total AA were greater during the first 24 h and also before and 2 h after feeding in PT than in T. The ratio of plasma BCAA to the aromatic AA (Tyr and Phe) was greatest in TC, followed by T, and least in PT. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in MLD and MM was greater in PT and T than in TC. The mRNA abundance of muscle-specific ligases FBXO32 (F-box only protein 32) in the 3 different skeletal muscles and TRIM63 (tripartite motif containing 63) in MLD was greater in PT and T than in TC; in MM, TRIM63 mRNA was greatest in PT. The mRNA for BCKDHA and BCKDHB (the α and β polypeptide of branched-chain α-keto acid dehydrogenase) in kidney fat was elevated in PT and T compared with TC, suggesting a possible enhancement of BCAA oxidation as energy source to cover the energetic and nutritional postnatal demands in PT and T in a starved state. The increased abundances of mTOR-associated signaling factors and muscle-specific ligase mRNA indicate a greater rate of protein turnover in muscles of PT and T in a starved state. Elevated plasma concentrations of several AA may result from enhanced muscle proteolysis and impaired conversion to glucose in the liver of PT calves.