Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Protein Expression and Functional Relevance of Efflux and Uptake Drug Transporters at the Blood-Brain Barrier of Human Brain and Glioblastoma.

The knowledge of transporter protein expression and function at the human blood-brain barrier (BBB) is critical to prediction of drug BBB penetration and design of strategies for improving drug delivery to the brain or brain tumor. This study determined absolute transporter protein abundances in isolated microvessels of human normal brain (N = 30), glioblastoma (N = 47), rat (N = 10) and mouse brain (N = 10), and cell membranes of MDCKII cell lines, using targeted proteomics. In glioblastoma microvessels, efflux transporters (ABCB1 and ABCG2), monocarboxylate transporter 1 (MCT1), glucose transporter 1 (GLUT1), sodium-potassium pump (Na/K ATPase), and Claudin-5 protein levels were significantly reduced, while large neutral amino acid transporter 1 (LAT1) was increased and GLU3 remained the same, as compared with human normal brain microvessels. ABCC4, OATP1A2, OATP2B1, and OAT3 were undetectable in microvessels of both human brain and glioblastoma. Species difference in BBB transporter abundances was noted. Cellular permeability experiments and modeling simulations suggested that not a single apical uptake transporter but a vectorial transport system consisting of an apical uptake transporter and basolateral efflux mechanism was required for efficient delivery of poor transmembrane permeability drugs from the blood to brain.

The ubiquitin specific protease USP34 protects the ubiquitin ligase gp78 from proteasomal degradation.

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.

Dissection of the Role of VIMP in Endoplasmic Reticulum-Associated Degradation of CFTRΔF508.

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important quality control mechanism that eliminates misfolded proteins from the ER. The Derlin-1/VCP/VIMP protein complex plays an essential role in ERAD. Although the roles of Derlin-1 and VCP are relatively clear, the functional activity of VIMP in ERAD remains to be understood. Here we investigate the role of VIMP in the degradation of CFTRΔF508, a cystic fibrosis transmembrane conductance regulator (CFTR) mutant known to be a substrate of ERAD. Overexpression of VIMP markedly enhances the degradation of CFTRΔF508, whereas knockdown of VIMP increases its half-life. We demonstrate that VIMP is associated with CFTRΔF508 and the RNF5 E3 ubiquitin ligase (also known as RMA1). Thus, VIMP not only forms a complex with Derlin-1 and VCP, but may also participate in recruiting substrates and E3 ubiquitin ligases. We further show that blocking CFTRΔF508 degradation by knockdown of VIMP substantially augments the effect of VX809, a drug that allows a fraction of CFTRΔF508 to fold properly and mobilize from ER to cell surface for normal functioning. This study provides insight into the role of VIMP in ERAD and presents a potential target for the treatment of cystic fibrosis patients carrying the CFTRΔF508 mutation.

SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTßR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTßR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

Quantitative and Mechanistic Understanding of AZD1775 Penetration across Human Blood-Brain Barrier in Glioblastoma Patients Using an IVIVE-PBPK Modeling Approach.

Purpose: AZD1775, a first-in-class, small-molecule inhibitor of the Wee1 tyrosine kinase, is under evaluation as a potential chemo- and radiosensitizer for treating glioblastoma. This study was to prospectively, quantitatively, and mechanistically investigate the penetration of AZD1775 across the human blood-brain barrier (BBB).Experimental Design: AZD1775 plasma and tumor pharmacokinetics were evaluated in 20 patients with glioblastoma. The drug metabolism, transcellular passive permeability, and interactions with efflux and uptake transporters were determined using human derived in vitro systems. A whole-body physiologically based pharmacokinetic (PBPK) model integrated with a four-compartment permeability-limited brain model was developed for predicting the kinetics of AZD1775 BBB penetration and assessing the factors modulating this process.Results: AZD1775 exhibited good tumor penetration in patients with glioblastoma, with the unbound tumor-to-plasma concentration ratio ranging from 1.3 to 24.4 (median, 3.2). It was a substrate for ABCB1, ABCG2, and OATP1A2, but not for OATP2B1 or OAT3. AZD1775 transcellular passive permeability and active efflux clearance across MDCKII-ABCB1 or MDCKII-ABCG2 cell monolayers were dependent on the basolateral pH. The PBPK model well predicted observed drug plasma and tumor concentrations in patients. The extent and rate of drug BBB penetration were influenced by BBB integrity, efflux and uptake active transporter activity, and drug binding to brain tissue.Conclusions: In the relatively acidic tumor microenvironment where ABCB1/ABCG2 transporter-mediated efflux clearance is reduced, OATP1A2-mediated active uptake becomes dominant, driving AZD1775 penetration into brain tumor. Variations in the brain tumor regional pH, transporter expression/activity, and BBB integrity collectively contribute to the heterogeneity of AZD1775 penetration into brain tumors. Clin Cancer Res; 23(24); 7454-66. ©2017 AACRSee related commentary by Peer et al., p. 7437.

Rapidly Translated Polypeptides Are Preferred Substrates for Cotranslational Protein Degradation.

Nascent polypeptides are degraded by the proteasome concurrently with their synthesis on the ribosome. This process, called cotranslational protein degradation (CTPD), has been observed for years, but the underlying mechanisms remain poorly understood. Equally unclear are the identities of cellular proteins genuinely subjected to CTPD. Here we report the identification of CTPD substrates in the yeast Saccharomyces cerevisiae via a quantitative proteomic analysis. We compared the abundance of individual ribosome-bound nascent chains between a wild type strain and a mutant defective in CTPD. Of 1,422 proteins acquired from the proteomic analysis, 289 species are efficient CTPD substrates, with >30% of their nascent chains degraded cotranslationally. We found that proteins involved in translation, ribosome biogenesis, nuclear transport, and amino acid metabolism are more likely to be targeted for CTPD. There is a strong correlation between CTPD and the translation efficiency. CTPD occurs preferentially to rapidly translated polypeptides. CTPD is also influenced by the protein sequence length; longer polypeptides are more susceptible to CTPD. In addition, proteins with N-terminal disorder have a higher probability of being degraded cotranslationally. Interestingly, the CTPD efficiency is not related to the half-lives of mature proteins. These results for the first time indicate an inverse correlation between CTPD and cotranslational folding on a proteome scale. The implications of this study with respect to the physiological significance of CTPD are discussed.

Gp78, an E3 ubiquitin ligase acts as a gatekeeper suppressing nonalcoholic steatohepatitis (NASH) and liver cancer.

Nonalcoholic steatohepatitis (NASH) is related to metabolic dysregulation and the perturbation of endoplasmic reticulum (ER) homeostasis that frequently develops into hepatocellular carcinoma (HCC). Gp78 is E3 ligase, which regulates endoplasmic reticulum-associated degradation (ERAD) by ubiquitinylation of misfolded ER proteins. Here, we report that upon ageing (12 months), gp78-/- mice developed obesity, recapitulating age-related human NASH. Liver histology of gp78-/- mice revealed typical steatosis, hepatic inflammation and fibrosis, followed by progression to hepatocellular tumors. Acute ER stress revealed that loss of gp78 results in up regulation of unfolded protein response (UPR) pathways and SREBP-1 regulating de novo lipogenesis, responsible for fatty liver. Tissue array of human hepatocellular carcinoma (HCC) demonstrated that the expression of gp78 was inversely correlated with clinical grades of cancer. Here, we have described the generation of the first preclinical experimental model system which spontaneously develops age-related NASH and HCC, linking ERAD to hepatosteatosis, cirrhosis, and cancer. It suggests that gp78 is a regulator of normal liver homeostasis and a tumor suppressor in human liver.

Dyclonine enhances the cytotoxic effect of proteasome inhibitor bortezomib in multiple myeloma cells.

The proteasome has become an important target for cancer therapy with the approval of bortezomib for the treatment of relapsed/refractory multiple myeloma (MM). However, numerous patients with MM do not respond to bortezomib and those responding initially often acquire resistance. Recent clinical studies have also demonstrated that bortezomib is also inefficacious in the treatment of other types of cancer. Therefore, it is imperative to develop novel approaches and agents for proteasome-targeting cancer therapy. In the present study, it was revealed that dyclonine, a major component of the cough droplets Sucrets, markedly enhances the cytotoxic effects of bortezomib and minimizes drug resistance in MM cells. It was demonstrated that a combination of bortezomib and dyclonine markedly induced apoptosis of MM cells. The present study suggests a novel therapeutic use of an over­the­counter medicine for the treatment of MM.

Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases.

gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.

Autocrine motility factor modulates EGF-mediated invasion signaling.

Autocrine motility factor (AMF) enhances invasion by breast cancer cells, but how its secretion and effector signaling are controlled in the tumor microenvironment is not fully understood. In this study, we investigated these issues with a chimeric AMF that is secreted at high levels through a canonical endoplasmic reticulum (ER)/Golgi pathway. Using this tool, we found that AMF enhances tumor cell motility by activating AKT/ERK, altering actin organization, and stimulating ß-catenin/TCF and activating protein 1 transcription. EGF enhanced secretion of AMF through its casein kinase II-mediated phosphorylation. RNA interference-mediated attenuation of AMF expression inhibited EGF-induced invasion by suppressing extracellular signal-regulated kinase signaling. Conversely, exogenous AMF overcame the inhibitory effect of EGF receptor inhibitor gefitinib on invasive motility by activating HER2 signaling. Taken together, our findings show how AMF modulates EGF-induced invasion while affecting acquired resistance to cytotoxic drugs in the tumor microenvironment.

Nuclear import factor Srp1 and its associated protein Sts1 couple ribosome-bound nascent polypeptides to proteasomes for cotranslational degradation.

Cotranslational protein degradation plays an important role in protein quality control and proteostasis. Although ubiquitylation has been suggested to signal cotranslational degradation of nascent polypeptides, cotranslational ubiquitylation occurs at a low level, suggesting the existence of an alternative route for delivery of nascent polypeptides to the proteasome. Here we report that the nuclear import factor Srp1 (also known as importin α or karyopherin α) is required for ubiquitin-independent cotranslational degradation of the transcription factor Rpn4. We further demonstrate that cotranslational protein degradation is generally impaired in the srp1-49 mutant. Srp1 binds nascent polypeptides emerging from the ribosome. The association of proteasomes with polysomes is weakened in srp1-49. The interaction between Srp1 and the proteasome is mediated by Sts1, a multicopy suppressor of srp1-49. The srp1-49 and sts1-2 mutants are hypersensitive to stressors that promote protein misfolding, underscoring the physiological function of Srp1 and Sts1 in degradation of misfolded nascent polypeptides. This study unveils a previously unknown role for Srp1 and Sts1 in cotranslational protein degradation and suggests a novel model whereby Srp1 and Sts1 cooperate to couple proteasomes to ribosome-bound nascent polypeptides.

The CCAAT box-binding transcription factor NF-Y regulates basal expression of human proteasome genes.

Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.

Structure, assembly and homeostatic regulation of the 26S proteasome.

The 26S proteasome is the major protease responsible for degradation of regulatory and abnormal proteins in the cell. Proteasomal degradation controls many cellular processes including, but not limited to, cell cycle control, transcription, DNA repair, apoptosis, quality control and antigen presentation. To elucidate how the proteasome is regulated is crucial to our understanding of the molecular details of proteasomal degradation and its functions in diverse biological pathways. In this article, I will highlight recent advances in understanding the proteasome structure and assembly and the regulation of proteasome gene expression. The implications of these new developments in cancer therapy will also be discussed.

Feedback regulation of proteasome gene expression and its implications in cancer therapy.

Proteasomal protein degradation is one of the major regulatory mechanisms in the cell. Aberrant proteasome activity is directly related to the pathogenesis of many human diseases including cancers. How proteasome homeostasis is controlled is a fundamental question toward our understanding of proteasome dysregulation in cancer cells. The recent discovery of the Rpn4-proteasome negative feedback circuit provides mechanistic insight into the regulation of proteasome gene expression. This finding also has important implications in cancer therapy that uses small molecule inhibitors to target the proteasome.

Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells.

Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors.

Diminished feedback regulation of proteasome expression and resistance to proteasome inhibitors in breast cancer cells.

Clinical trials with proteasome inhibitor Bortezomib (also named Velcade or PS-341) has shown promising results for some cancers. However, other types of cancers including breast cancer do not respond well to Bortezomib. To understand the cause of the drug resistance, we compared the regulation of proteasome expression and the sensitivity to proteasome inhibitors between human breast cancer cells and nontumorigenic mammary epithelial cells. We found that, while the endogenous expression level is much higher, the potential of feedback expression in response to proteasome inhibitors is much lower in the breast cancer cells. Furthermore, the breast cancer cells are much more resistant to proteasome inhibitors compared to the nontumorigenic mammary epithelial cells. Biochemical analysis showed that the pathway of Bortezomib-induced apoptosis is apparently defective in the breast cancer cells. Together, these results provide an explanation for the inefficacy of Bortezomib in the clinical trials for breast cancer patients. The likelihood of combination therapy with Bortezomib and other anti-cancer agents for breast cancer is also discussed.

Proteasomal degradation of RPN4 via two distinct mechanisms, ubiquitin-dependent and -independent.

Substrates of the ubiquitin system are degraded by the 26 S proteasome, a complex protease consisting of at least 32 different subunits. Recent studies showed that RPN4 (also named SON1 and UFD5) is a transcriptional activator required for normal expression of the Saccharomyces cerevisiae proteasome genes. Interestingly, RPN4 is extremely short-lived and degraded by the 26 S proteasome, establishing a feedback circuit that controls the homeostatic abundance of the 26 S proteasome. The mechanism underlying the degradation of RPN4, however, remains unclear. Here we demonstrate that the proteasomal degradation of RPN4 is mediated by two independent degradation signals (degron). One degron leads to ubiquitylation on internal lysine(s), whereas the other is independent of ubiquitylation. Stabilization of RPN4 requires inhibition of internal ubiquitylation and inactivation of the ubiquitin-independent degron. RPN4 represents the first proteasomal substrate in S. cerevisiae that can be degraded through ubiquitylation or without prior ubiquitylation. This finding makes it possible to use both yeast genetics and biochemical analysis to investigate the mechanism of ubiquitin-independent proteolysis.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.