Structural and functional consequences of c-N-Ras constitutively associated with intact mitochondria

AbstractWe demonstrate that both c-N-Ras and c-K(B)-Ras are constitutively associated with purified mitochondria. c-K(B)-Ras is associated with the mitochondrial outer membrane, and c-N-Ras is associated with both the outer membrane and inner mitochondrial compartments. The mitochondrial morphology is abnormal in both c-N-Ras negative and K-Ras negative cells. Normal mitochondrial morphology was restored by targeting N-Ras to both the inner and outer mitochondrial compartments, or by ectopically expressing c-K(B)-Ras. Impaired mitochondrial function can result in increased CHOP and NFκB activity, typical for a retrograde signaling response. Both are constitutively elevated in the N-Ras negative cells, but not in the K-Ras negative background, and are restored by c-N-Ras targeted exclusively to the inner mitochondrial compartment. Surprisingly, both targeting and the ability to functionally reduce retrograde transcriptional activity were found to be independent of c-N-Ras farnesylation. Overall, these data demonstrate for the first time a (1) farnesylation independent function for c-N-Ras and (2) that N-Ras within the inner mitochondrial compartment is an essential component of the retrograde signaling system between the mitochondria and nucleus

Early highly effective versus escalation treatment approaches in relapsing multiple sclerosis

Treatment decisions in multiple sclerosis are complex given the large number of disease-modifying therapies with diverse safety and efficacy profiles. The importance of early treatment has been recognised but how intensively to treat at onset is not known. Substantial variability exists in treatment selection with weak clinical trial evidence to guide initial treatment choices. Decision-making is made more complicated by variable tolerance for risk of side-effects and inability to accurately predict treatment response. Whether to use moderately effective and safe medications with escalation as needed, or to use higher efficacy medications from the outset, is a key question in clinical practice. Clinical trials in patients with relapsing multiple sclerosis have focused on pairwise comparisons but the effectiveness of different treatment approaches has not been tested. Future pragmatic randomised clinical trials and observational studies will help to inform more rational selection of initial therapies and improve the quality of life of patients with relapsing multiple sclerosis

ATP modulates PTEN subcellular localization in multiple cancer cell lines

The tumour suppressor gene PTEN plays an important somatic role in both hereditary and sporadic breast carcinogenesis. While the role of PTEN's lipid phosphatase activity, as a negative regulator of the cytoplasmic phosphatidylinositol-3-kinase/Akt pathway is well known, it is now well established that PTEN exists and functions in the nucleus. Multiple mechanisms of regulating PTEN's subcellular localization have been reported. However none are ubiquitous across multiple cancer cell lines and tissue types. We show here that adenosine triphosphate (ATP) regulates PTEN subcellular localization in a variety of different cancer cell lines, including those derived from breast, colon and thyroid carcinomas. Cells deficient in ATP show an increased level of nuclear PTEN protein. This increase in PTEN is reversed when cells are supplemented with ATP, ADP or AMP. In contrast, the addition of the non-hydrolyzable analogue ATPγS, did not reverse nuclear PTEN protein levels in all the cell types tested. To our knowledge, this is the first report that describes a regulation of PTEN subcellular localization that is not specific to one cell line or tissue type, but appears to be common across a variety of cell lineages

Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function

Germline and somatic PTEN mutations are found in Cowden syndrome (CS) and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of CS patients and sporadic colorectal carcinomas and comparative aminoacid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type (WT) or mutant PTEN to bind ATP was assessed by ATP–agarose-binding assays. Subcellular fractionation, western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60–73; c.122–136). In contrast to WT PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant PTEN that does not bind ATP efficiently. We also show that CS patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTEN's ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations

Determining the effectiveness of early intensive versus escalation approaches for the treatment of relapsing-remitting multiple sclerosis: The DELIVER-MS study protocol

Multiple Sclerosis (MS) is a common cause of neurological disability among young adults and has a high economic burden. Currently there are 18 disease modifying agents for relapsing MS, which were tested in clinical trials versus placebo or an active comparator in a pairwise manner. However, there is currently no consensus on the fundamental principles of treatment approach and initial therapy selection. These factors result in variable use of disease modifying therapies. Here we describe the study protocol for Determining the Effectiveness of earLy Intensive Versus Escalation approaches for the Treatment of Relapsing-remitting Multiple Sclerosis (DELIVER-MS). The main objective of the study is to determine whether an early highly effective treatment approach, defined as use of one of four monoclonal antibodies as initial therapy, is more effective than an escalation treatment approach (any other approved medication as initial therapy with subsequent escalation to higher efficacy treatments guided by radiological and clinical evaluation). The primary endpoint of the study is reduction in normalized brain volume loss from baseline visit to month 36 visit using MRI. Brain volume loss was selected as the best short-term predictor of long-term clinical disability. A total of 400 participants will be randomized 1:1 using minimization to account for age and sex by site, and 400 will be enrolled into a parallel observational cohort. The study results will help guide overall treatment philosophy and will have important implications for patient choice, clinical practice, and treatment access

Collaborative International Research in Clinical and Longitudinal Experience Study in NMOSD.

Objective: To develop a resource of systematically collected, longitudinal clinical data and biospecimens for assisting in the investigation into neuromyelitis optica spectrum disorder (NMOSD) epidemiology, pathogenesis, and treatment. Methods: To illustrate its research-enabling purpose, epidemiologic patterns and disease phenotypes were assessed among enrolled subjects, including age at disease onset, annualized relapse rate (ARR), and time between the first and second attacks. Results: As of December 2017, the Collaborative International Research in Clinical and Longitudinal Experience Study (CIRCLES) had enrolled more than 1,000 participants, of whom 77.5% of the NMOSD cases and 71.7% of the controls continue in active follow-up. Consanguineous relatives of patients with NMOSD represented 43.6% of the control cohort. Of the 599 active cases with complete data, 84% were female, and 76% were anti-AQP4 seropositive. The majority were white/Caucasian (52.6%), whereas blacks/African Americans accounted for 23.5%, Hispanics/Latinos 12.4%, and Asians accounted for 9.0%. The median age at disease onset was 38.4 years, with a median ARR of 0.5. Seropositive cases were older at disease onset, more likely to be black/African American or Hispanic/Latino, and more likely to be female. Conclusions: Collectively, the CIRCLES experience to date demonstrates this study to be a useful and readily accessible resource to facilitate accelerating solutions for patients with NMOSD

ATP modulates PTEN subcellular localization in

multiple cancer cell line

Tyk2 Tyrosine Kinase Expression Is Required for the Maintenance of Mitochondrial Respiration in Primary Pro-B Lymphocytes

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-α/β). Although Tyk2(−/−) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(−/−) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-β. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-β. Finally, Tyk2(−/−) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration