Ubiquitin-proteasome Proteolytic Pathway Research Articles

LncRNA H19 Regulates Breast Cancer DNA Damage Response and Sensitivity to PARP Inhibitors via Binding to ILF2.

Although DNA damage repair plays a critical role in cancer chemotherapy, the function of lncRNAs in this process remains largely unclear. In this study, in silico screening identified H19 as an lncRNA that potentially plays a role in DNA damage response and sensitivity to PARP inhibitors. Increased expression of H19 is correlated with disease progression and with a poor prognosis in breast cancer. In breast cancer cells, forced expression of H19 promotes DNA damage repair and resistance to PARP inhibition, whereas H19 depletion diminishes DNA damage repair and increases sensitivity to PARP inhibitors. H19 exerted its functional roles via direct interaction with ILF2 in the cell nucleus. H19 and ILF2 increased BRCA1 stability via the ubiquitin-proteasome proteolytic pathway via the H19- and ILF2-regulated BRCA1 ubiquitin ligases HUWE1 and UBE2T. In summary, this study has identified a novel mechanism to promote BRCA1-deficiency in breast cancer cells. Therefore, targeting the H19/ILF2/BRCA1 axis might modulate therapeutic approaches in breast cancer.

Stability of Dengue 2 Nonstructural Glycoprotein 1 (NS1) Is Affected by the Nature of Basic Residue at Position NS1-324.

Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the β-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.

C188-9, a specific inhibitor of STAT3 signaling, prevents thermal burn-induced skeletal muscle wasting in mice.

Burn injury is the leading cause of death and disability worldwide and places a tremendous economic burden on society. Systemic inflammatory responses induced by thermal burn injury can cause muscle wasting, a severe involuntary loss of skeletal muscle that adversely affects the survival and functional outcomes of these patients. Currently, no pharmacological interventions are available for the treatment of thermal burn-induced skeletal muscle wasting. Elevated levels of inflammatory cytokines, such as interleukin-6 (IL-6), are important hallmarks of severe burn injury. The levels of signal transducer and activator of transcription 3 (STAT3)-a downstream component of IL-6 inflammatory signaling-are elevated with muscle wasting in various pro-catabolic conditions, and STAT3 has been implicated in the regulation of skeletal muscle atrophy. Here, we tested the effects of the STAT3-specific signaling inhibitor C188-9 on thermal burn injury-induced skeletal muscle wasting in vivo and on C2C12 myotube atrophy in vitro after the administration of plasma from burn model mice. In mice, thermal burn injury severity dependently increased IL-6 in the plasma and tibialis anterior muscles and activated the STAT3 (increased ratio of phospho-STAT3/STAT3) and ubiquitin-proteasome proteolytic pathways (increased Atrogin-1/MAFbx and MuRF1). These effects resulted in skeletal muscle atrophy and reduced grip strength. In murine C2C12 myotubes, plasma from burn mice activated the same inflammatory and proteolytic pathways, leading to myotube atrophy. In mice with burn injury, the intraperitoneal injection of C188-9 (50mg/kg) reduced activation of the STAT3 and ubiquitin-proteasome proteolytic pathways, reversed skeletal muscle atrophy, and increased grip strength. Similarly, pretreatment of murine C2C12 myotubes with C188-9 (10µM) reduced activation of the same inflammatory and proteolytic pathways, and ameliorated myotube atrophy induced by plasma taken from burn model mice. Collectively, these results indicate that pharmacological inhibition of STAT3 signaling may be a novel therapeutic strategy for thermal burn-induced skeletal muscle wasting.

Irisin promotes the proliferation and tenogenic differentiation of rat tendon-derived stem/progenitor cells via activating YAP/TAZ

Tendinopathy is a common tendon disorder characterized by pain, swelling, and dysfunction. Current evidence has demonstrated that the depletion of stem cell pool and non-tenogenic differentiation of tendon-derived stem/progenitor cells (TSPCs) might account for the pathogenesis of tendinopathy. FNDC5/Irisin, as a novel exercise-induced myokine, is proved to be involved in the exercise-induced protective effects on musculoskeletal disorders. However, whether irisin can affect TSPCs fate is still unknown. To ascertain the roles of irisin on the proliferation and tenogenic differentiation of TSPCs, rat TSPCs were isolated and incubated with irisin. Cell viability, phenotypic changes, and related signaling pathways were evaluated by CCK-8 assay, colony formation assay, real-time PCR, Western blot, immunofluorescence, and proteasome activity assay. We found that irisin treatment increased the proliferative and colony-forming abilities, and promoted the tenogenic differentiation of TSPCs by upregulating the expression of YAP/TAZ. In conclusion, our work showed for the first time that irisin promotes the proliferation and tenogenic differentiation of rat TSPCs in vitro by activating YAP/TAZ, and the process was associated with a ubiquitin-proteasome proteolytic pathway. In conclusion, irisin and agents targeting YAP/TAZ may be promising therapeutic options for tendinopathy.

JNK/Itch Axis Mediates the Lipopolysaccharide-Induced Ubiquitin-Proteasome-Dependent Degradation of Ferritin Light Chain in Murine Macrophage Cells.

Ferritin, which is composed of a heavy chain and a light chain, plays a critical role in maintaining iron homeostasis by sequestering iron. The ferritin light chain (FTL) is responsible for the stability of the ferritin complex. We have previously shown that overexpression of FTL decreases the levels of the labile iron pool (LIP) and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-treated murine macrophage cells. The protein level of FTL was downregulated by LPS within a short treatment period. However, the mechanism underlying the LPS-induced changes in the FTL levels is not known. In the present study, we report that LPS induces the ubiquitin-proteasome-dependent degradation of FTL and that the mechanism of LPS-induced FTL degradation involves the JNK/Itch axis. We found that LPS downregulates the protein and mRNA levels of FTL in a time-dependent manner. The proteasome inhibitor MG-132 significantly reverses the LPS-induced decrease in FTL. Furthermore, we observed that LPS treatment cannot cause ubiquitination of the lysine site (K105 and K144) mutant of FTL. Interestingly, LPS-mediated ubiquitin-dependent degradation of FTL is significantly inhibited by the JNK-specific inhibitor SP600125. Moreover, LPS could upregulate the protein level of E3 ubiquitin ligase Itch, a substrate of JNK kinases. Immunoprecipitation analyses revealed an increase in the association of FTL with Itch, a substrate of JNK kinases, in response to LPS stimulation. SP600125 decreased LPS-induced Itch upregulation. Taken together, these results suggest that LPS stimulation leads to the degradation of FTL through the ubiquitin-proteasome proteolytic pathway, and this FTL degradation is mediated by the JNK/Itch axis in murinemacrophage cells.

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

KDM5c Promotes Colon Cancer Cell Proliferation Through the FBXW7-c-Jun Regulatory Axis.

KDM5c is a histone demethylase that specifically demethylates trimethylated and dimethylated H3 Lys-4 to play a central role in transcriptional repression. C-Jun is a proto-oncogene and promotes cell proliferation when ectopically accumulated, but can be ubiquitinated by SCF (FBXW7), leading to its degradation. FBXW7 is an E3 ubiquitin ligase of c-Jun, and exhibits carcinostasis in colon cancer. Here, we report that overexpression of KDM5c in human colon cancer cells results in attenuated FBXW7 transcription and accumulated c-Jun protein, leading to increased proliferation of colon cancer cells. We show that overexpression of KDM5c can result in increased c-Jun protein levels and decreased ubiquitin levels, with no significant change in mRNA levels of c-Jun. KDM5c overexpression blocks the ubiquitin-proteasome proteolytic pathway of c-Jun by down-regulating the expression of FBXW7. KDM5c down-regulation of FBXW7 occurs by demethylation of H3K4me3 at TSS and downstream of the FBXW7 gene. And interaction of KDM5c with H3K4me3 downstream of FBXW7 gene may be followed by recruitment of DNMT3b to methylate the spatially close CpG island located near the FBXW7 TSS. This methylation represses FBXW7 gene expression, which can reduce c-Jun degradation via the ubiquitin-proteasome pathway. TCGA database analysis revealed high expression of KDM5c in colon cancer tissues. KDM5c expression in colon cancer was correlated with poor overall survival of patients in the first 7 years. Data from TCGA showed that high expression of KDM5c was correlated with high DNA methylation of the FBXW7 gene, but was not positively correlated with methylation of the Jun gene. These results suggest that KDM5c regulation of colon cell proliferation is mainly mediated by the KDM5c-FBXW7-c-Jun axis.

Regulation of Autophagy in Chick Skeletal Muscle: Effect of mTOR Inhibition.

Autophagy in the skeletal muscle increases under catabolic conditions resulting in muscle atrophy. This study investigated the effect of inhibition of mechanistic target of rapamycin (mTOR) on autophagy in chick skeletal muscle. We examined the effects of Torin1, an mTOR inhibitor, on autophagy. Chick myotubes were incubated with Torin1 (100 nM) for 3 h. It was observed that Torin1 inhibited the phosphorylation of AKT (Ser473), p70 ribosomal S6 kinase 1 (S6K1, Thr389), S6 ribosomal protein (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46), which are used for measurement of mTOR activity. Torin1 significantly (P< 0.01) increased the LC3-II/LC3-I ratio, an index for autophagosome formation, while it did not influence the expression of autophagy-related genes (LC3B, GABARAPL1, and ATG12). In addition, Torin1 increased atrogin-1/MAFbx (a muscle-specific ubiquitin ligase) mRNA expression. Fasting for 24 h inhibited the phosphorylation of AKT (Ser473), S6K1 (Ther389), S6 ribosomal protein (Ser235/236), and 4E-BP1 (Thr37/46) in chick skeletal muscle and significantly (P<0.01) increased the LC3-II/LC3-I ratio. Fasting also increased GABARAPL1 and atrogin-1/MAFbx mRNA expression but not LC3B or ATG12 mRNA expression. These results indicate that mTOR signaling regulates autophagy and the ubiquitin-proteasome proteolytic pathway in chick skeletal muscle.

Ixazomib Maintenance to Prevent Alloreactivity and Provide Anti-Malignancy Effects Following Nonmyeloablative T-Replete Haploidentical Peripheral Blood Stem Cell Transplantation with Post-Transplant Cyclophosphamide

Inhibition of the ubiquitin-proteasome proteolytic pathway results in extensive immunomodulatory effects, augmenting NK cell cytotoxicity while inhibiting aspects of T-, B- and dendritic cell function. To this end, short course bortezomib has been utilized post-transplant to decrease the risk of graft-versus-host disease (GVHD) while preserving graft-versus-malignancy (GVM) effects (Koreth, Haematologica 2018). We performed a phase II study examining the effects of ixazomib for GVHD prophylaxis (up to 12 cycles), in addition to post-transplant cyclophosphamide and tacrolimus, following the original Baltimore nonmyeloablative haploidentical transplant (HIDT) regimen. We hypothesized that substituting ixazomib for mycophenolate would provide acceptable GVHD control while potentially augmenting GVM. Ixazomib was started on day +5 at a dose of 4mg on days 1, 8, 15 of a 28-day cycle, with dose reductions allowed in future cycles for toxicity and mycophenolate, used for conventional post-HIDT GVHD prophylaxis, was eliminated. Donors were selected to maximize NK alloreactivity (i.e. B/x KIR haplotype). Twenty-five patients were enrolled, median age 62 (range 35-77) years, with acute leukemia [4], MDS [7], NHL/HL/CLL [8] and myeloma [6]. Comorbidity index (HCT-CI) was ≥3 in 68%. Donor KIR haplotype was B/x and A/A in 18 and 7 patients respectively. After a median follow-up of 33.5 months (range 9.2-52.8), 19 patients survive with an estimated 1- and 3-year overall survival of 84% and 74% respectively. Engraftment occurred in all patients with no secondary graft failure. Median time to ANC&gt;500 and PLT&gt;20 was 16 and 29 days respectively. CMV reactivation was frequent occurring in 88% of CMV-seropositive patients, however there was no cases of CMV disease. Infectious mortality occurred in 3 patients (disseminated adenovirus, HHV6 encephalitis and fungal pneumonia). The 3-yr cumulative incidence of non-relapse mortality (NRM) was 12% but was significantly higher when the donor possessed the more inhibitory KIR A/A vs. stimulatory B/x haplotype (43% vs. 0%). Relapse incidence at 1- and 3-years was 24% and 44%. Cumulative incidence of Grade 3-4 acute GVHD at day +180 was 8%, while 2-year moderate-to-severe chronic GVHD occurred in 21%. Hematologic and cutaneous toxicities possibly related to ixazomib were common, resulting in dose reductions and treatment interruptions in most patients. Median number of ixazomib cycles was 3 (range 1-12), with only one patient receiving all 12 planned cycles. Rash, at least possibly related to ixazomib, was particularly frequent occurring in 19 (76%) patients at a median day +32 (range, 17-139) following transplant. Reasons for early ixazomib discontinuation included rash [9], cytopenias [7], relapse/progression [4], GVHD [2], diarrhea [1] and arthralgias [1]. In conclusion, the substitution of ixazomib for mycophenolate post-HIDT is feasible and results in reliable engraftment with low rates of clinically significant GVHD and NRM, without any appreciable effect on relapse protection. The occurrence of rash in the first several months in three quarters of patients was unexpected and made it difficult to distinguish drug toxicity from low-grade skin-only acute GVHD. The finding that Infectious mortality occurred exclusively in recipients of KIR A/A haplotype donors is interesting and requires further study. Figure Disclosures Solh: Celgene: Speakers Bureau; Amgen: Speakers Bureau; ADC Therapeutics: Research Funding. OffLabel Disclosure: Use of ixazomib for GVHD prophlaxis following haploidentical transplantation.

Beta-Hydroxy-Beta-Methyl Butyrate (HMB): From Experimental Data to Clinical Evidence in Sarcopenia.

β-hydroxy-β-methylbutyrate (HMB) is a metabolite derived from leucine and its ketoacid alpha- ketoisocaproate. Leucine has a role in regulating protein synthesis in muscle cells, and HMB seems to be a key active metabolite in such regulation. HMB has been shown to modulate muscle protein degradation by inhibiting the ubiquitin-proteasome proteolytic pathway, to up-regulate protein synthesis via the mTOR pathway, and to stabilize cell membranes via the rate limiting enzyme to cholesterol synthesis HMG- coenzyme A reductase. It can also decrease cell apoptosis, therefore improving cell survival; and increase proliferation and differentiation of muscle stem cell, via the MAPK/ERK and PI3K/Akt pathways. HMB is widely used as an ergogenic supplement by athletes and bodybuilders, usually combined with exercise training, to increase muscle mass and strength. Some studies have explored the role of HMB in chronic diseases associated with muscle wasting (cancer, acquired immunodeficiency syndrome, chronic obstructive pulmonary disease). This review focuses on the role of HMB in the management of sarcopenia (age or disease-related loss of muscle mass and function) in older persons. A small number of studies have shown increases in lean (muscle) mass and some muscle function and physical performance parameters in older people with or without resistance exercise, and preservation of muscle mass during bed rest. However, heterogeneous methodological approaches preclude solid conclusions, and more studies are needed to confirm the role of HMB as a promising agent to treat sarcopenia.

Transcriptome profiling of Issatchenkia orientalis under ethanol stress

Issatchenkia orientalis, a non-Saccharomyces yeast that can resist a wide variety of environmental stresses, has potential use in winemaking and bioethanol production. Little is known about gene expression or the physiology of I. orientalis under ethanol stress. In this study, high-throughput RNA sequencing was used to investigate the transcriptome profile of I. orientalis in response to ethanol. 502 gene transcripts were differentially expressed, of which 451 were more abundant, and 51 less abundant, in cells subjected to 4 h of ethanol stress (10% v/v). Annotation and statistical analyses suggest that multiple genes involved in ergosterol biosynthesis, trehalose metabolism, and stress response are differentially expressed under these conditions. The up-regulation of molecular chaperones HSP90 and HSP70, and also genes associated with the ubiquitin–proteasome proteolytic pathway suggests that ethanol stress may cause aggregation of misfolded proteins. Finally, ethanol stress in I. orientalis appears to have a nitrogen starvation effect, and many genes involved in nutrient uptake were up-regulated.

Regulation of Autophagy in Chick Myotubes: Effects of Insulin, Insulin-Like Growth Factor-I, and Amino Acids.

Autophagy, an intracellular bulk protein degradation system in skeletal muscle, is increased under catabolic conditions resulting in muscle atrophy. This study aimed to investigate the effects of insulin, insulin-like growth factor (IGF)-I, and amino acids on autophagy (LC3-II content and expression of autophagy-related genes) in chick myotubes. Chick myotubes were incubated with insulin (1 µg/ml), IGF-I (100 ng/ml), and amino acids for 3 h. The LC3-II content, an index of autophagosome formation, and mRNA expression of LC3B and GABARAPL1 were significantly decreased by insulin. The LC3-II content, but not mRNA expression of autophagy-related genes, was also significantly decreased by IGF-I. The LC3-II content and LC3B mRNA level were also significantly decreased by amino acids. The mRNA expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, was also significantly decreased by insulin, IGF-I, and amino acids in chick myotubes. These results indicated that insulin, IGF-I, and amino acids regulate autophagy as well as the ubiquitin–proteasome proteolytic pathway in chick myotubes.

Investigating the underlying mechanism of Saccharomyces cerevisiae in response to ethanol stress employing RNA-seq analysis

Saccharomyces cerevisiae has been widely used for wine fermentation and bio-fuels production. A S. cerevisiae strain Sc131 isolated from tropical fruit shows good fermentation properties and ethanol tolerance, exhibiting significant potential in Chinese bayberry wine fermentation. In this study, RNA-sequence and RT-qPCR was used to investigate the transcriptome profile of Sc131 in response to ethanol stress. Scanning Electron Microscopy were carried out to observe surface morphology of yeast cells. Totally, 937 genes were identified differential expressed, including 587 up-regulated and 350 down-regulated genes, after 4-h ethanol stress (10% v/v). Transcriptomic analysis revealed that, most genes involved in regulating filamentous growth or pseudohyphal growth were significantly up-regulated in response to ethanol stress. The complex protein quality control machineries, Hsp90/Hsp70 and Hsp104/Hsp70/Hsp40 based chaperone system combining with ubiquitin-proteasome proteolytic pathway were both activated to recognize and degrade misfolding proteins. Genes related to biosynthesis and metabolism of two well-known stress-responsive substances trehalose and ergosterol were generally up-regulated, while genes associated with amino acids biosynthesis and metabolism processes were differentially expressed. Moreover, thiamine was also important in response to ethanol stress. This research may promote the potential applications of Sc131 in the fermentation of Chinese bayberry wine.

UCP2 Regulates Embryonic Neurogenesis via ROS-Mediated Yap Alternation in the Developing Neocortex.

Mitochondrial metabolism is a fundamental process in tissue development. How this process play functions in embryonic neurogenesis remains largely unknown. Here, we show that mitochondrial uncoupling protein 2 (UCP2) regulates the embryonic neurogenesis by inhibiting the production of reactive oxygen species (ROS), which affect the proliferation of progenitors. In the embryonic brains of UCP2 knockdown or condition knockout mice, the proliferation of progenitors is significantly increased, while the differentiation of progenitors is reduced. Furthermore, we identify that Yap is the response protein of UCP2-mediated ROS production. When UCP2 is inactive, the production of ROS is increased. The amount of Yap protein is increased as Yap degradation through ubiquitin-proteasome proteolytic pathway is decreased. The defect caused by UCP2 depression can be rescued by Yap downregulation. Collectively, our results demonstrate that UCP2 regulates embryonic neurogenesis through ROS-mediated Yap alternation, thus shedding new sight on mitochondrial metabolism involved in embryonic neurogenesis. Stem Cells 2017;35:1479-1492.

Abstract P1-08-09: Aurora kinase-A protein stability is negatively regulated by eEF1α2 and PTEN in breast cancer: Prognostic and therapeutic implications

Abstract Background: The AURKA gene encoding Aurora kinase-A (Aurora-A) protein is localized on chromosome 20q13 that is frequently amplified and overexpressed across multiple cancer types correlating with patient prognosis. Aurora-A plays a pivotal role in faithful segregation of chromosomes and normal progression of mitosis, peaking at G2/M followed by degradation at the end of mitosis by APC/C (Cdh1). However, regulation of Aurora-A protein stability in human cancer cells is not well elucidated. Here, we show that Aurora-A is targeted for ubiquitination and degradation by SCF complex involving eEF1α2 and PTEN in human breast cancer cells. Methods: Using a panel of breast cancer cell lines, as in vitro models, the eEF1α2 was knocked down or ectopically expressed to test the stability of Aurora-A protein. For in vivo models, tissue micro arrays of human breast cancer were immunostained for Aurora-A and eEF1α2 expression and categorized values were statistically tested by Chi-squared test. In addition, the public breast cancer dataset (Transbig) was used to predict breast cancer prognosis by Kaplan-Meier survival analysis. Results: In breast cancer cell lines and patient samples, an eEF1α2 non-expressing group showed a trend of higher Aurora-A expression than eEF1α2 expressing group, whose trend was significant in patient samples (P&amp;lt;0.05). Knocking down of eEF1α2 enhanced Aurora-A protein stability. In contrast, ectopic expression of eEF1α2 dramatically decreased Aurora-A protein stability. Inhibition of proteasome activity by MG132 could restore the Aurora-A protein in eEF1α2 expressing cells. Biochemical assays showed the direct binding between eEF1α2 and Aurora-A, and eEF1α2 dependent ubiquitination of Aurora-A. Inverse correlation of the expression levels of the two proteins was also observed throug the cell cycle, with eEF1α2 levels being high from G1 through G2 phases while Aurora-A expression peaked from G2/M phase through cytokinesis. Taken together, these findings highlight eEF1α2 as a novel negative regulator destabilizing Aurora-A through ubiquitin-proteasome proteolytic pathway. Further, mechanistic studies revealed that eEF1α2 enhanced the interaction of SCF E3 ubiquitin ligase complex protein; FBXW7 and Cul1, with Aurora-A. In line with this scenario, knocking down of Cul1 increased Aurora-A level. Since PTEN loss was reported to stabilize Aurora-A through inhibiting SCF complex, we tested the significance of PTEN loss in our model. Knocking down of PTEN further stabilized Aurora-A suggesting an independent role of PTEN from eEF1α2 in destabilizing Aurora-A. When eEF1α2 expressing cells were treated with AKT inhibitor, Aurora-A was destabilized with enhanced bindings between Aurora-A and FBXW7/Cul1. Lastly, low PTEN expression correlated with poor prognosis of Aurora-A over expressing breast cancer patients (P&amp;lt;0.01). Conclusions: Aurora-A overexpression in human breast cancer cells may be associated with loss of eEF1α2 and PTEN due to reduced interaction of SCF with Aurora-A. Findings indicate significant prognostic and therapeutic implications of altered expression of eEF1α2/PTEN/Aurora-A pathways among Aurora-A subset of breast cancer patients. Citation Format: Treekitkarnmongkol W, Kai K, Katayama H, Tian W, Rodriguez-Canales J, Sahin AA, Sen S. Aurora kinase-A protein stability is negatively regulated by eEF1α2 and PTEN in breast cancer: Prognostic and therapeutic implications [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-09.

Mass Spectrometry: A Technique of Many Faces.

Protein complexes form the critical foundation for a wide range of biological process, however understanding the intricate details of their activities is often challenging. In this review we describe how mass spectrometry plays a key role in the analysis of protein assemblies and the cellular pathways which they are involved in. Specifically, we discuss how the versatility of mass spectrometric approaches provides unprecedented information on multiple levels. We demonstrate this on the ubiquitin-proteasome proteolytic pathway, a process that is responsible for protein turnover. We follow the various steps of this degradation route and illustrate the different mass spectrometry workflows that were applied for elucidating molecular information. Overall, this review aims to stimulate the integrated use of multiple mass spectrometry approaches for analyzing complex biological systems.

A novel in vitro model of sarcopenia using BubR1 hypomorphic C2C12 myoblasts.

Sarcopenia is the age-related loss of skeletal muscle mass and function with adverse outcomes that include physical disability, poor quality of life, and death. The detailed molecular mechanisms remain unknown. An in vitro muscle atrophy model is needed to enable mechanistic studies. To create such a model, we employed BubR1 insufficiency which causes premature ageing in mice. Using C2C12 cells, a recognized in vitro model of the skeletal muscle cell, we obtained the BubR1 hypomorphic C2C12 (C2C12BKD) cells by using shRNA. The resulting C2C12BKD cells displayed several characteristics of the sarcopenic muscle cell. In C2C12BKD cells, formation of myotubes, assessed by analysis of fusion index, was markedly reduced as was the expression of myogenin and MyoD, two marker genes for myogenesis. Moreover, the cells showed increased expression of the muscle-specific ubiquitin ligases Atrogin-1 and MuRF-1, indicating increased protein degradation through the ubiquitin-proteasome dependent proteolytic pathway. These results suggest that C2C12BKD cells are potentially useful as a novel in vitro model of sarcopenia.

Physiology of the Alzheimer’s disease

Alzheimer’s disease is a disease that is resulted from increased plasma osmolality both the excessive consumption of animal-based proteins and reduction of sodium intake, that resulted to increase plasma osmolality. When we are exposed to high animal-based protein diets throughout life, we gradually lose extracellular sodium and the body cannot retain water, resulting in a gradual rise of plasma osmolality. When the neuronal cells of the central nervous system are exposed to high osmolality stress, they produce of phosphorylated tau, APP, and pathologic beta amyloid protein peptides. The BACE 1 protein which influences the cleavage of amyloid precursor proteins (APP) and affects the production of beta amyloid protein peptides, is also increased in a hyperosmotic stress. When pathologic beta amyloid protein peptides are produced, they are degraded by the ubiquitin proteasome proteolytic pathway, and only then are the neurotoxic effects on the central nervous system manifested, leading to Alzheimer’s disease.

Modulatory Effects of Bortezomib on Host Immune Cell Functions

Bortezomib is an inhibitor of the ubiquitin-proteasome proteolytic pathway responsible for intracellular protein turnover. Cellular proteins controlled by this pathway represent a diverse group of potential therapeutic targets, particularly in cancer cells, which exploit this proteasomal pathway to promote their growth and diminish apoptosis. Along with inhibiting the proteasome and thus sensitizing tumor cells to apoptosis, bortezomib may also have multiple effects on the host immune responses. This review summarizes the effects that bortezomib may play on immune cell subsets in various disease states in modifying lymphocyte receptors, ligands, the expression of various cytokines and chemokines and their downstream signaling. We also propose steps that can be taken to refine combinatorial strategies that include bortezomib to improve current immunotherapeutic approaches.

Molecular mechanisms of skeletal muscle atrophy in a mouse model of cerebral ischemia.

Loss of muscle mass and function is a severe complication in patients with stroke that contributes to promoting physical inactivity and disability. The deleterious consequences of skeletal muscle mass loss underline the necessity to identity the molecular mechanisms involved in skeletal muscle atrophy after cerebral ischemia. Transient focal cerebral ischemia (60 minutes) was induced by occlusion of the right middle cerebral artery in C57BL/6J male mice. Skeletal muscles were removed 3 days later and analyzed for the regulation of critical determinants of muscle mass homeostasis (Akt/mammalian target of rapamycin pathway, myostatin-Smad2/3 and bone morphogenetic protein-Smad1/5/8 signaling pathways, ubiquitin-proteasome and autophagy-lysosome proteolytic pathways). Cerebral ischemia induced severe sensorimotor deficits associated with muscle mass loss of the paretic limbs. Mechanistically, cerebral ischemia repressed Akt/mammalian target of rapamycin pathway and increased expression of key players of ubiquitin-proteasome pathway (MuRF1 [muscle RING finger-1], MAFbx [muscle atrophy F-box], Musa1 [muscle ubiquitin ligase of SCF complex in atrophy-1]), together with a marked increase in myostatin expression, in both paretic and nonparetic skeletal muscles. The Smad1/5/8 pathway was also activated. Our data fit with a model in which a repression of Akt/mammalian target of rapamycin pathway and an increase in the expression of key players of ubiquitin-proteasome pathway are critically involved in skeletal muscle atrophy after cerebral ischemia. Cerebral ischemia also caused an activation of bone morphogenetic protein-Smad1/5/8 signaling pathway, suggesting that compensatory mechanisms are also concomitantly activated to limit the extent of skeletal muscle atrophy.