Kosj Yamoah

Nedd8 on cullin: building an expressway to protein destruction

This review summarizes recent advances concerning the Nedd8 regulatory pathway in four areas. One, substantial progress has been made in delineating the role of cullin family proteins, the only known substrates of the Nedd8 modification system. Cullins are molecular scaffolds responsible for assembling the ROC1/Rbx1 RING-based E3 ubiquitin ligases, of which several play a direct role in tumorigenesis. Two, a large body of work has helped elucidate the molecular details underlying the Nedd8 modification reaction, which results in covalent conjugation of a Nedd8 moiety onto a conserved cullin lysine residue. Three, studies using a variety of genetic model systems have established an essential role for Nedd8 in cell cycle control and in embryogenesis by upregulating the activities of cullin-based E3 ligases. In vitro experiments have revealed a direct role for Nedd8 in activating ubiquitination. Construction of a model of the ROC1/Rbx1-CUL1-Nedd8 structure suggests a mechanism by which the cullin-linked Nedd8 may assist the neighboring ROC1/Rbx1 in landing and positioning the E2 conjugating enzyme for the ubiquitin transfer reaction. Finally, increasing evidence indicates that removal of Nedd8 from its cullin targets, by the action of COP9 Signalosome and possibly other proteases, plays a significant role in the regulation of cullin-mediated proteolysis.

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin‐mediated degradation of important signaling intermediates, including β‐catenin and the NF‐κB inhibitor IκB‐α. Ubiquitination of these proteins as well as others is catalyzed by the SCFβTrCP ubiquitin ligase, which itself requires regulated modification of the cullin‐1 subunit by the ubiquitin‐like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8‐conjugating enzyme, resulting in complete but transient loss of cullin‐1 neddylation and consequent effects on NF‐κB and β‐catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin–proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail

SCF (Skp1·CUL1·F-box protein·ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1s stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD·ROC1 interaction, markedly increased the ability of SCFβTrCP2 to promote IκBα polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD·ROC1 interaction.

Novel Biomarker Signature That May Predict Aggressive Disease in African American Men With Prostate Cancer

PURPOSE We studied the ethnicity-specific expression of prostate cancer (PC) -associated biomarkers to evaluate whether genetic/biologic factors affect ethnic disparities in PC pathogenesis and disease progression. PATIENTS AND METHODS A total of 154 African American (AA) and 243 European American (EA) patients from four medical centers were matched according to the Cancer of the Prostate Risk Assessment postsurgical score within each institution. The distribution of mRNA expression levels of 20 validated biomarkers reported to be associated with PC initiation and progression was compared with ethnicity using false discovery rate, adjusted Wilcoxon-Mann-Whitney, and logistic regression models. A conditional logistic regression model was used to evaluate the interaction between ethnicity and biomarkers for predicting clinicopathologic outcomes. RESULTS Of the 20 biomarkers examined, six showed statistically significant differential expression in AA compared with EA men in one or more statistical models. These include ERG (P < .001), AMACR (P < .001), SPINK1 (P = .001), NKX3-1 (P = .03), GOLM1 (P = .03), and androgen receptor (P = .04). Dysregulation of AMACR (P = .036), ERG (P = .036), FOXP1 (P = .041), and GSTP1 (P = .049) as well as loss-of-function mutations for tumor suppressors NKX3-1 (P = .025) and RB1 (P = .037) predicted risk of pathologic T3 disease in an ethnicity-dependent manner. Dysregulation of GOLM1 (P = .037), SRD5A2 (P = .023), and MKi67 (P = .023) predicted clinical outcomes, including 3-year biochemical recurrence and metastasis at 5 years. A greater proportion of AA men than EA men had triple-negative (ERG-negative/ETS-negative/SPINK1-negative) disease (51% v 35%; P = .002). CONCLUSION We have identified a subset of PC biomarkers that predict the risk of clinicopathologic outcomes in an ethnicity-dependent manner. These biomarkers may explain in part the biologic contribution to ethnic disparity in PC outcomes between EA and AA men.

Bone loss in thyroid disease: role of low TSH and high thyroid hormone.

Abstract:  More than 10% of postmenopausal women in the United States receive thyroid hormone replacement therapy and up to 20% of these women are over‐replaced inducing subclinical hyperthyroidism. Because hyperthyroidism and post menopausal osteoporosis overlap in women of advancing age, it is urgent to understand the effect of thyroid hormone excess on bone. We can now provide results that not thyroid hormones but also TSH itself has an equally important role to play in bone remodeling.

Human Cdc34 employs distinct sites to coordinate attachment of ubiquitin to a substrate and assembly of polyubiquitin chains.

ABSTRACT The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IκBα while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IκBα, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34E108A/E112A abolished both poly- and monoubiquitination of IκBα. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2s N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.

African American men with low-grade prostate cancer have increased disease recurrence after prostatectomy compared with Caucasian men.

PURPOSE To explore whether disparities in outcomes exist between African American (AA) and Caucasian (CS) men with low-grade prostate cancer and similar cancer of the prostate risk assessment-postsurgery (CAPRA-S) features following prostatectomy (RP). METHODS The overall cohort consisted of 1,265 men (234 AA and 1,031 CS) who met the National comprehensive cancer network criteria for low- to intermediate-risk prostate cancer and underwent RP between 1990 and 2012. We first evaluated whether clinical factors were associated with adverse pathologic outcomes and freedom from biochemical failure (FFbF) using the entire cohort. Next, we studied a subset of 705 men (112 AA and 593 CS) who had pathologic Gleason score≤6 (low-grade disease). Using this cohort, we determined whether race affected FFbF in men with RP-proven low-grade disease and similar CAPRA-S scores. RESULTS With a median follow-up time of 27 months, the overall 7-year FFbF rate was 86% vs. 79% in CS and AA men, respectively (P = 0.035). There was no significant difference in one or more adverse pathologic features between CS vs. AA men (27% vs. 31%; P = 0.35) or CAPRA-S score (P = 0.28). In the subset analysis of patients with low-grade disease, AA race was associated with worse FFbF outcomes (P = 0.002). Furthermore, AA race was a significant predictor of FFbF in men with low-grade disease (hazard ratio = 2.01, 95% CI: 1.08-3.72; P = 0.029). CONCLUSIONS AA race is a predictor of worse FFbF outcomes in men with low-grade disease after RP. These results suggest that a subset of AA men with low-grade disease may benefit from more aggressive treatment.

Impact of Race on Biochemical Disease Recurrence After Prostate Brachytherapy

Understanding racial differences in disease presentation and response to therapy is necessary for the effective treatment and control of prostate cancer. In this study, the authors examined the influence of race on biochemical disease‐free survival (BDFS) among men who received prostate brachytherapy.

Radiation Therapy Intensification for Solid Tumors: A Systematic Review of Randomized Trials

PURPOSE To systematically review the outcomes of randomized trials testing radiation therapy (RT) intensification, including both dose escalation and/or the use of altered fractionation, as a strategy to improve disease control for a number of malignancies. METHODS AND MATERIALS We performed a literature search to identify randomized trials testing RT intensification for cancers of the central nervous system, head and neck, breast, lung, esophagus, rectum, and prostate. Findings were described qualitatively. Where adequate data were available, pooled estimates for the effect of RT intensification on local control (LC) or overall survival (OS) were obtained using the inverse variance method. RESULTS In primary central nervous system tumors, esophageal cancer, and rectal cancer, randomized trials have not demonstrated that RT intensification improves clinical outcomes. In breast cancer and prostate cancer, dose escalation has been shown to improve LC or biochemical disease control but not OS. Radiation therapy intensification may improve LC and OS in head and neck and lung cancers, but these benefits have generally been limited to studies that did not incorporate concurrent chemotherapy. CONCLUSIONS In randomized trials, the benefits of RT intensification have largely been restricted to trials in which concurrent chemotherapy was not used. Novel strategies to optimize the incorporation of RT in the multimodality treatment of solid tumors should be explored.