ABSTRACT
The activity of cullin-RING type ubiquitination E3 ligases is regulated by neddylation, a process analogous to ubiquitination that culminates in covalent attachment of the ubiquitin-like protein Nedd8 to cullins. As a component of the E3 for neddylation, SCCRO/DCUN1D1 plays a key regulatory role in neddylation and, consequently, cullin-RING ligase activity. The essential contribution of SCCRO to neddylation is to promote nuclear translocation of the cullin-ROC1 complex. The presence of a myristoyl sequence in SCCRO3, one of four SCCRO paralogues present in humans that localizes to the membrane, raises questions about its function in neddylation. We found that although SCCRO3 binds to CAND1, cullins, and ROC1, it does not efficiently bind to Ubc12, promote cullin neddylation, or conform to the reaction processivity paradigms, suggesting that SCCRO3 does not have E3 activity. Expression of SCCRO3 inhibits SCCRO-promoted neddylation by sequestering cullins to the membrane, thereby blocking its nuclear translocation. Moreover, SCCRO3 inhibits SCCRO transforming activity. The inhibitory effects of SCCRO3 on SCCRO-promoted neddylation and transformation require both an intact myristoyl sequence and PONY domain, confirming that membrane localization and binding to cullins are required for in vivo functions. Taken together, our findings suggest that SCCRO3 functions as a tumor suppressor by antagonizing the neddylation activity of SCCRO.
Subject(s)
Carcinogenesis , Cell Cycle Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitins/metabolism , Active Transport, Cell Nucleus , Carrier Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Cullin Proteins/metabolism , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins , NEDD8 Protein , Protein Structure, Tertiary , Proteins , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/geneticsABSTRACT
PURPOSE: To determine mechanisms by which SCCRO5 (aka DCUN1D5) promotes oncogenesis. EXPERIMENTAL DESIGN: SCCRO5 mRNA and protein expression were assessed in 203 randomly selected primary cancer tissue samples, matched histologically normal tissues, and cell lines by use of real-time PCR and Western blot analysis. SCCRO5 overexpression was correlated with survival. The effect of SCCRO5 knockdown on viability was assessed in selected cancer cell lines. Structure-function studies were performed to determine the SCCRO5 residues required for binding to the neddylation components, for neddylation-promoting activity, and for transformation. RESULTS: In oral and lung squamous cell carcinomas, SCCRO5 mRNA levels corresponded with protein levels and overexpression correlated with decreased disease-specific survival. Knockdown of SCCRO5 by RNAi resulted in a selective decrease in the viability of cancer cells with high endogenous levels, suggesting the presence of oncogene addiction. SCCRO5 promoted cullin neddylation while maintaining conserved reaction processivity paradigms involved in ubiquitin and ubiquitin-like protein conjugation, establishing it as a component of the neddylation E3. Neddylation activities in vitro required the potentiating of neddylation (PONY) domain but not the nuclear localization sequence (NLS) domain. In contrast, both the NLS domain and the PONY domain were required for transformation of NIH-3T3 cells. CONCLUSIONS: Our data suggest that SCCRO5 has oncogenic potential that requires its function as a component of the neddylation E3. Neddylation activity and nuclear localization of SCCRO5 are important for its in vivo function.
Subject(s)
Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Peptide Synthases/genetics , Peptide Synthases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Cell Proliferation , Cullin Proteins/metabolism , Disease Progression , Gene Expression , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/mortality , Phenotype , Protein Binding , Protein Transport , Ubiquitins/metabolismABSTRACT
PURPOSE: To evaluate the use of selective vs. non-selective cyclo-oxygenase inhibitors (COXIs) for the treatment of chronic non-infectious, non-necrotizing scleritis and episcleritis. METHODS: Sixty-nine patients with scleritis and episcleritis treated for ≥2 months with COXIs were included. Outcome measures were rates of inflammation control, corticosteroid sparing, and COXI discontinuation, as well as side effects. RESULTS: Initial inflammation control was achieved in 78-81% of scleritis patients and 73-80% episcleritis patients on COXIs (p > .05). Rates of steroid sparing after 4-24 months of consecutive treatment were similar. Gastrointestinal side effects were observed in 1 (2.7%) patient on selective vs. 3 (9.4%) patients on non-selective COXIs (p = .33). Overall discontinuation rate was 6/37 (16%) for selective vs. 7/32 (22%) for non-selective COXIs (p = .76). CONCLUSIONS: Selective vs. non-selective COXIs were equally efficacious for the treatment of chronic non-infectious, non-necrotizing scleritis and episcleritis. Prospective studies to elucidate potential differences in side effect profiles may be warranted.
Subject(s)
Cyclooxygenase Inhibitors/therapeutic use , Scleritis/drug therapy , Adult , Aged , Chronic Disease , Female , Gastrointestinal Diseases/chemically induced , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Retrospective Studies , Treatment Outcome , Visual Acuity/drug effectsABSTRACT
PURPOSE: To determine the effectiveness and corticosteroid (CS) sparing capabilities of mycophenolate mofetil (MMF) in the treatment of chronic non-infectious, non-necrotizing scleritis. METHODS: A retrospective chart review of patients treated for scleritis at the Institute of Ophthalmology and Visual Science at New Jersey Medical School was performed. Only those patients taking MMF for greater than or equal to six consecutive months were included. Main outcome measures were rate of inflammation control, CS, and MMF discontinuation, as well as visual acuity and side effects. RESULTS: Twenty-two patients (32 eyes) were included in the study. Mean ± SD age was 53.5 ± 13.3 years. Twenty (91%) patients had previously failed some form of immunomodulatory therapy. After 6, 12, 18, and 24 months of consecutive MMF treatment, 91-100% of patients achieved inflammation control. Mean time to resolution of inflammation was 2.8 months, while mean duration of inflammation control was 14.8 months. CS sparing was achieved in 100% of patients at each time point, with mean starting CS dose decreased by 91% at final visit. Vision was stable or improved in 24 (75%) eyes. Fourteen (64%) patients reported side effects including leucopenia (n = 7), gastrointestinal upset (n = 4), abnormal liver function tests (n = 3), and abnormal renal function tests (n = 2). None required hospitalization or medical treatment. Four (18%) patients discontinued MMF due to side effects (n = 3) and treatment ineffectiveness (n = 1). CONCLUSION: MMF is an effective and well-tolerated therapy that can successfully reduce inflammation and decrease CS use in the treatment of chronic non-infectious, non-necrotizing scleritis.
