Proteasome-mediated Turnover Research Articles

Chloroplast elongation factors break the growth-immunity trade-off by simultaneously promoting yield and defence.

Chloroplasts regulate plant development and immunity. Here we report that potato chloroplast elongation factors StTuA and StTuB, targeted by Phytophthora infestans RXLR effector Pi22926, positively regulate immunity and growth. Plants expressing Pi22926, or silenced for TuA/B, show increased P. infestans susceptibility and decreased photosynthesis, plant growth and tuber yield. By contrast, StTuA/B overexpression reduces susceptibility, elevates chloroplast-derived reactive oxygen species production and increases photosynthesis and potato tuber yield by enhancing chloroplast protein translation. Another plant target of Pi22926, StMAP3Kβ2, interacts with StTuB, phosphorylating it to promote its translocation into chloroplasts. However, Pi22926 attenuates StTuB association with StMAP3Kβ2 and phosphorylation. This reduces StTuB translocation into chloroplasts, leading to its proteasome-mediated turnover in the cytoplasm. We uncover new mechanisms by which a pathogen effector inhibits immunity by disrupting key chloroplast functions. This work shows that StTuA/B break the growth-immunity trade-off, promoting both disease resistance and yield, revealing the enormous potential of chloroplast biology in crop breeding.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

NIPSNAP1 directs dual mechanisms to restrain senescence in cancer cells

BackgroundAlthough the executive pathways of senescence are known, the underlying control mechanisms are diverse and not fully understood, particularly how cancer cells avoid triggering senescence despite experiencing exacerbated stress conditions within the tumor microenvironment.MethodsMass spectrometry (MS)-based proteomic screening was used to identify differentially regulated genes in serum-starved hepatocellular carcinoma cells and RNAi employed to determine knockdown phenotypes of prioritized genes. Thereafter, gene function was investigated using cell proliferation assays (colony-formation, CCK-8, Edu incorporation and cell cycle) together with cellular senescence assays (SA-β-gal, SAHF and SASP). Gene overexpression and knockdown techniques were applied to examine mRNA and protein regulation in combination with luciferase reporter and proteasome degradation assays, respectively. Flow cytometry was applied to detect changes in cellular reactive oxygen species (ROS) and in vivo gene function examined using a xenograft model.ResultsAmong the genes induced by serum deprivation, NIPSNAP1 was selected for investigation. Subsequent experiments revealed that NIPSNAP1 promotes cancer cell proliferation and inhibits P27-dependent induction of senescence via dual mechanisms. Firstly, NIPSNAP1 maintains the levels of c-Myc by sequestering the E3 ubiquitin ligase FBXL14 to prevent the proteasome-mediated turnover of c-Myc. Intriguingly, NIPSNAP1 levels are restrained by transcriptional repression mediated by c-Myc-Miz1, with repression lifted in response to serum withdrawal, thus identifying feedback regulation between NIPSNAP1 and c-Myc. Secondly, NIPSNAP1 was shown to modulate ROS levels by promoting interactions between the deacetylase SIRT3 and superoxide dismutase 2 (SOD2). Consequent activation of SOD2 serves to maintain cellular ROS levels below the critical levels required to induce cell cycle arrest and senescence. Importantly, the actions of NIPSNAP1 in promoting cancer cell proliferation and preventing senescence were recapitulated in vivo using xenograft models.ConclusionsTogether, these findings reveal NIPSNAP1 as an important mediator of c-Myc function and a negative regulator of cellular senescence. These findings also provide a theoretical basis for cancer therapy where targeting NIPSNAP1 invokes cellular senescence.

Abstract 2327: Novel role of NAC1 in regulating tumor stem cells and their interaction with immune cells in triple-negative breast cancer

Abstract One of the important features of triple-negative breast cancer (TNBC) is the enrichment of stem-like cells that contribute substantially to the malignant phenotype. Nucleus accumbens associated protein 1 (NAC1), a unique member of the BTB-POZ family, is implicated in tumor development and progression of various types of cancer. The objective of this study was to test the hypothesis that NAC1 plays a critical role in maintaining stemness of TNBC. We found that TNBC cell lines, MDA-MB-231 and HCC-1806, had high NAC1 expressions compared to normal. Hematoxylin-eosin staining of 450 human breast cancer tissues revealed high NAC1 expressions in different stages of breast tumor development. Depletion of NAC1 in TNBC cells decreased their mammosphere formation, migration and invasion abilities, and altered their EMT phenotype. Canonical TNBC stemness markers CD44 and ALDH1A1 were downregulated in TNBC cells subjected to NAC1 depletion. Down-regulation of NAC1 also decreased aldolase activity. RNA sequencing uncovered the enrichment of JAK/STAT3, PI3K/AKT, WNT, stemness and tumor progression-associated genes in TNBC with depletion of NAC1. IHC analysis revealed increased expression of phospho-STAT3 (Y705), β-catenin, PDK1, cyclin D1, cMYC, IL-6 and phosphorylated PTEN in TNBC cells in support of the sequencing data. Immunoprecipitation assays demonstrated the physical association of NAC1 with STAT3 and CD44 and the pulse-chase experiments showed that the proteasome-mediated turnover of STAT3 was faster in the NAC1-depleted cells than the control cells (half-life: 5hrs vs 24hrs, respectively). NAC1-depleted cells had a significantly reduced tumorigenic ability in vivo. TCGA immune cell infiltration analysis revealed that NAC1 expression had a positive correlation with myeloid-derived suppressor cells (MDSCs) infiltration. In vitro co-culture assays with Gr1/CD11b+ cells from NAC1 knockout mice with EO771 tumor cells showed increase of the stemness marker CD44 and aldolase activity, suggesting a role of NAC1 in modulating the stemness-immune interaction in tumor microenvironment. Further, we observed a significant increase of NAC1 in MDSCs from the tumor bearing mice compared to that of the tumor-free mice. The results of this study demonstrate a novel role of NAC1 in regulating tumor stem cells and their crosstalk with immune cells in the tumor microenvironment (TME) and suggest that targeting NAC1 may be exploited as a new approach to reverse immune-suppressive TME and to improve treatment for TNBC. Citation Format: Chrispus M. Ngule, Hami Hemati, Xingcong Ren, Emily Bachert, Xiaoqi Liu, Jin-Ming Yang. Novel role of NAC1 in regulating tumor stem cells and their interaction with immune cells in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2327.

CHMP2B regulates TDP-43 phosphorylation and cytotoxicity independent of autophagy via CK1.

The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.

Proteasomal degradation of human SERINC4: A potent host anti-HIV-1 factor that is antagonized by nef

The serine incorporator (SERINC) protein family has five paralogous members with 9–11 transmembrane domains. SERINC5 is a potent host restriction factor and antagonized by HIV-1 Nef and two other retroviral accessory proteins via the lysosomal degradation pathway. Here, we investigated human SERINC4 expression and antiviral mechanisms. Unlike its four paralogs, human SERINC4 is subjected to proteasome-mediated turnover, resulting in ~250-fold lower expression than SERINC5. However, when expression was normalized, human SERINC4 restricted HIV-1 replication as effectively as SERINC5, and SERINC4 was also antagonized by Nef via the lysosomal pathway. Although SERINC4 proteins are conserved within primates or rodents, their N-terminal regions are highly variable across species. Interestingly, unlike human SERINC4, murine SERINC4 was stably expressed but had a very poor antiviral activity. We created stable SERINC4 chimeras by replacing the N-terminal region and found that the 1–34 and 35–92 amino acids determine SERINC4 antiviral activity or protein expression, respectively. Using these chimeras, we demonstrate that SERINC4 is incorporated into HIV-1 virions and restricts Tier 1 HIV-1 more effectively than Tier 3 HIV-1. Importantly, SERINC4 increases HIV-1 sensitivity to broadly neutralizing antibodies. Thus, human SERINC4 strongly restricts HIV-1 replication when it is overexpressed, which reflects a potential antiviral activity of this gene product under physiological conditions.

The kinase activity of the channel-kinase protein TRPM7 regulates stability and localization of the TRPM7 channel in polarized epithelial cells.

The channel-kinase transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein with ion channel and kinase domains. The kinase activity of TRPM7 has been linked to the regulation of a broad range of cellular activities, but little is understood as to how the channel itself is regulated by its own kinase activity. Here, using several mammalian cell lines expressing WT TRPM7 or kinase-inactive variants, we discovered that compared with the cells expressing WT TRPM7, cells in which TRPM7's kinase activity was inactivated had faster degradation, elevated ubiquitination, and increased intracellular retention of the channel. Mutational analysis of TRPM7 autophosphorylation sites further revealed a role for Ser-1360 of TRPM7 as a key residue mediating both TRPM7 stability and intracellular trafficking. Additional trafficking roles were uncovered for Ser-1403 and Ser-1567, whose phosphorylation by TRPM7's kinase activity mediated the interaction of the channel with the signaling protein 14-3-3θ. In summary, our results point to a critical role for TRPM7's kinase activity in regulating proteasome-mediated turnover of the TRPM7 channel and controlling its cellular localization in polarized epithelial cells. Overall, these findings improve our understanding of the significance of TRPM7's kinase activity for functional regulation of its channel activity.

UXT Is a LOX-PP Interacting Protein That Modulates Estrogen Receptor Alpha Activity in Breast Cancer Cells.

The lysyl oxidase proenzyme propeptide region (LOX-PP) is a tumor suppressor protein whose mechanism of action is not completely understood. Here, the Ubiquitously expressed Transcript (UXT) was identified in a yeast two-hybrid assay with LOX-PP as bait and confirmed as a novel LOX-PP associating protein. UXT, a prefoldin-like protein, is ubiquitous in human and mouse. Since UXT modulates androgen receptor transcriptional activity in prostate cancer, we studied its role in breast cancer. Breast tumors and derived cell lines overexpressed UXT. UXT was able to associate with the estrogen receptor alpha (ER) and decrease its transcriptional activity and target gene expression. Conversely, UXT knockdown increased ER element-dependent transcriptional activity. Ectopic LOX-PP relocalized UXT to the cytoplasm and decreased its stability. UXT ubiquitination and depletion in the presence of LOX-PP was rescued by a proteasomal inhibitor. In summary, proteasome-mediated turnover of UXT upon interaction with LOX-PP releases repression of ER transcriptional activity. J. Cell. Biochem. 118: 2347-2356, 2017. © 2017 Wiley Periodicals, Inc.

Establishment of a suite of assays that support the discovery of proteasome stimulators

BackgroundThe proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing. MethodsThe cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates. ResultsTo demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold. ConclusionAssays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides. General significanceA suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.

Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses.

The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.

Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.

Drosophila Morgue Associates with SkpA and Polyubiquitin In Vivo

Morgue is a unique ubiquitination protein that influences programmed cell death and circadian rhythms in Drosophila. We have found that over-expression of wild-type Morgue results in organismal lethality. This over-expression phenotype was used as the basis for an in vivo functional assay to investigate the importance of the Morgue zinc finger, F box, Ubiquitin E2 Conjugase Variant (UEV) domain, and active site Glycine residue. Removal of the zinc finger or UEV domain reduced Morgue’s ability to induce lethality and enhance cell death. In contrast, lack of the F box as well as several different substitutions of the active site Glycine did not alter Morgue-induced lethality or cell death enhancement. To further characterize Morgue functions, a Flag:Morgue protein was used to isolate Morgue-associated proteins from whole adult Drosophila. Mass spectrometry analysis of the Morgue-associated proteins identified SkpA as well as a ubiquitin multimer. The identification of SkpA is consistent with previous in vitro studies and further suggests Morgue acts in an SCF-type ubiquitin E3 ligase complex. The identification of poly-ubiquitin was unexpected and this interaction had not been previously identified. The associated poly-ubiquitin was found to exhibit a Lys-48 topology, consistent with distinct functions of Morgue in proteasome-mediated protein turnover. Multiple regions of Morgue were subsequently shown to be required for poly-ubiquitin binding. Overall, Morgue is a novel multi-functional ubiquitin-binding protein.

Induction of p53, p21 and apoptosis by silencing the NF90/NF45 complex in human papilloma virus-transformed cervical carcinoma cells

The heterodimeric nuclear factor 90/nuclear factor 45 complex (NF90/NF45) binds nucleic acids and is a multifunctional regulator of gene expression. Here we report that depletion of NF90/NF45 restores the expression of the p53 and p21 proteins in cervical carcinoma cells infected with high-risk human papillomaviruses (HPV). Knockdown of either NF90 or NF45 by RNA interference led to greatly elevated levels of p53 and p21 proteins in HPV-derived HeLa and SiHa cells, but not in other cancerous or normal cell lines. In HeLa cells, p21 mRNA increased concomitantly but the level of p53 mRNA was unaffected. RNA interference directed against p53 prevented the induction of both proteins. These results indicated that the up-regulation of p21 is due to p53-dependent transcription, whereas p53 is regulated post-transcriptionally. Proteasome-mediated turnover of p53 is accelerated by the HPV E6 and cellular E6AP proteins. We therefore examined the hypothesis that this pathway is regulated by NF90/NF45. Indeed, depletion of NF90 attenuated the expression of E6 RNA and inhibited transcription from the HPV early promoter, revealing a new role for NF90/NF45 in HPV gene expression. The transcription inhibition was largely independent of the reduction of P-TEFb levels caused by NF90 depletion. Consistent with p53 derepression, NF90/NF45-depleted HeLa cells displayed elevated PARP cleavage and susceptibility to camptothecin-induced apoptosis. We conclude that high-risk strains of HPV utilize the cellular NF90/NF45 complex for viral E6 expression in infected cervical carcinoma cell lines. Interference with NF90/NF45 function could assist in controlling cervical carcinoma.

Proteasome-Mediated Turnover of Arabidopsis MED25 Is Coupled to the Activation of FLOWERING LOCUS T Transcription

The Mediator complex is a greater than 1-megadalton complex, composed of about 30 subunits and found in most eukaryotes, whose main role is to transmit signals from DNA-bound transcription factors to RNA Polymerase II. The proteasome is emerging as an important regulator of transcription during both initiation and elongation. It is increasing the number of cases where the proteolysis of transcriptional activators by the proteasome activates their function. This counterintuitive phenomenon was called "activation by destruction." Here, we show that, in Arabidopsis (Arabidopsis thaliana), PHYTOCHROME AND FLOWERING TIME1 (PFT1), the MEDIATOR25 (MED25) subunit of the plant Mediator complex, is degraded by the proteasome and that proteasome-mediated PFT1 turnover is coupled to its role in stimulating the transcription of FLOWERING LOCUS T, the plant florigen, which is involved in the process of flowering induction. We further identify two novel RING-H2 proteins that target PFT1 for degradation. We show that MED25-BINDING RING-H2 PROTEIN1 (MBR1) and MBR2 bind to PFT1 in yeast (Saccharomyces cerevisiae) and in vitro, and they promote PFT1 degradation in vivo, in a RING-H2-dependent way, typical of E3 ubiquitin ligases. We further show that both MBR1 and MBR2 also promote flowering by PFT1-dependent and -independent mechanisms. Our findings extend the phenomenon of activation by destruction to a Mediator subunit, adding a new mechanism by which Mediator subunits may regulate downstream genes in specific pathways. Furthermore, we show that two novel RING-H2 proteins are involved in the destruction of PFT1, adding new players to this process in plants.

Functional linkage of Retinoblastoma protein family turnover and potency during mouse embryonic stem cell development

In embryonic stem (ES) cells, the Retinoblastoma family of proteins is held in a biochemically inactive state through robust cyclin/cdk‐mediated phosphorylation. However, at the onset of differentiation, diminished cyclin/cdk activity contributes to the establishment of functional RB/p107/p130 pathways and regulated cell cycle control. We report that the invocation of pocket protein function during ES cell differentiation is also correlated with decreased repressor abundance, suggesting that repressor potency is linked to turnover. Indeed, wild type p107 levels were diminished by pharmacological inhibition of cyclin/cdk activity while mutant p107 lacking an evolutionarily conserved instability element within its C‐terminal regulatory domain was refractory to cyclin/cdk modulation. Select amino acid substitutions within this instability element increased p107 abundance and phosphorylation, but crippled p107 repressor function. The instability elements of both p107 and p130 can direct proteasome‐mediated turnover when fused to heterologous reporter proteins indicating that these elements can function as autonomous degrons. Importantly, mutations within the p130 degron have been observed in human lung carcinoma patients indicating an important role for the regulation of degron function in human disease. This research was supported by the National Institutes of Health.

ISG15 disrupts cytoskeletal architecture and promotes motility in human breast cancer cells

The interferon-stimulated gene 15 (ISG15) pathway is highly elevated in breast cancer; however, very little is known about how the ISG15 pathway contributes to breast tumorigenesis. In the current study, using the gene disruption approach, we demonstrate that both ISG15 and UbcH8 (ISG15-specific conjugating enzyme) disrupt F-actin architecture and formation of focal adhesions in ZR-75-1 breast cancer cells. In addition, ISG15 and UbcH8 promote breast cancer cell migration. We also demonstrate that ISG15 inhibits ubiquitin/26S proteasome-mediated turnover of proteins implicated in tumor cell motility, invasion and metastasis. Together, our results suggest that the aberrant activation of the ISG15 pathway confers a motile phenotype to breast cancer cells by disrupting cell architecture and stabilizing proteins involved in cell motility, invasion and metastasis. Because the cellular architecture is conserved and the ISG15 pathway is constitutively activated in tumor cells of different lineages, it is reasonable to assume that our observations in breast cancer must hold true for many other tumors.

A Novel Role for ATM in Regulating Proteasome-Mediated Protein Degradation through Suppression of the ISG15 Conjugation Pathway

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein

Background Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG) represents ∼90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden.Methodology/Principal FindingsWe demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as ∼2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation.Conclusions/SignificanceThe retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only ∼30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.

Nuclear CDKs Drive Smad Transcriptional Activation and Turnover in BMP and TGF-β Pathways

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways.