Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.Transforming growth factor-β (TGF-β)¹ superfamily ligands, including TGF-βs, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-β superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000–8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in multiple organs, potentially leading to severe hemorrhages and strokes (4). Haploinsufficiency is the underlying cause of HHT, indicating that reduced levels of functional endoglin or ACVRL1 (ALK1) proteins predispose to endothelial dysfunction and AVMs (5). Although the mechanisms responsible for AVMs remain unclear, the elucidation of how members of the TGF-β superfamily and their molecular networks regulate vascular integrity is vital for future treatments of HHT.We have demonstrated that endoglin interacts with endothelial nitric oxide synthase (NOS3 or eNOS) and regulates its activation (2). NOS3 is a Ca⁺² and calmodulin-regulated enzyme that produces NO● in response to humoral and mechanical stimuli via dynamic interactions with various allosteric regulators such as heat shock protein 90 (HSP90). NOS3 is also regulated by dynamic changes in its phosphorylation status. For example, effects of the vascular endothelial growth factor (VEGF) on angiogenesis, vascular permeability and vasomotor tone are mediated in part through Akt-dependent phosphorylation of NOS3 Ser1177 and by increased NOS3-HSP90 association (6). Although phosphorylation of NOS3 Ser1177 is indicative of agonist-induced activation, it is preceded by dephosphorylation at Thr495. TGF-β1 and -β3 but not -β2 responses can sensitize NOS3 for activation by inducing dephosphorylation at Thr495, and therefore contribute to NOS3 activation and NO-dependent vasorelaxation (7). Endoglin regulates TGF-β1 and -β3 but not -β2 responses, and is required for their induction of NOS3 Thr495 dephosphorylation (7, 8).In the vascular endothelium of HHT patients and in Eng and Alk1 heterozygous mice, impaired association of NOS3 with HSP90 renders the enzyme uncoupled, causing production of superoxide (●O₂⁻) instead of NO● (2, 3, 9) and leading to endothelial damage. Interestingly, TGF-β1 and -β3 do not induce phosphorylation at NOS3 Ser1177, yet NOS3 activation in response to TGF-β1 is abolished in endoglin-deficient cells, impairing vasomotor function (3). ACVRL1 (or ALK1) also interacts with NOS3, and its reduced levels in endothelial cells similarly cause NOS3-derived oxidative stress (3, 9).In view of the crucial roles of endoglin and ACVRL1 in the development and maintenance of the normal vasculature and the definite contribution of their mutated state to HHT, we used the LUMIER high-throughput technology (10) to identify novel protein interactions and molecular networks for these predominantly endothelial receptors. We included TGFBR2 to further define TGF-β protein networks potentially important for vascular function, and attempt to distinguish the TGF-β networks from those associated with BMP9/BMP10 and mediated by ACVRL1 in a complex with BMPR2 and endoglin (11, 12).One of identified proteins interacting with all three receptors was protein phosphatase 2A (PP2A), implicated in multiple pathways. PP2A is a holoenzyme with one structural subunit (PPP2R1A or PPP2R1B) associated with one catalytic subunit (PPP2CA or PPP2CB) and one of 19 regulatory B subunits, the latter conferring specificity to the enzyme by recruiting interacting proteins (13, 14). Of interest, PP2A interacts with NOS3 to regulate Ser1177 phosphorylation and NO● production (15). However, the mechanisms governing recruitment of PP2A to NOS3 and the contribution of TGF-β/BMP receptor complexes are unknown. Recently, the human PPP2R2B gene coding for PPP2R2B protein (also known as PP2A-Bβ regulatory subunit) was mapped to chromosome 5q31-q32, in an interval in linkage disequilibrium with the HHT3 locus (16, 17). We now report that PPP2R2B interacts with the ACVRL1/TGFBR2/endoglin complex and that endoglin governs NOS3 phosphorylation and activation status by hindering PP2A access to NOS3 via the PPP2R2B subunit. Loss of endoglin leads to constitutive reduction in NOS3 phosphorylation and likely to changes in several networks with consequent endothelial dysfunction.     

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.     

Microtubules, MAPs and plant directional cell expansion

Plant microtubules (MTs) polymerize and depolymerize in a process termed dynamic instability. This allows the assembly, reorganization, and disassembly of at least four MT arrays throughout the cell cycle. The cortical MT array lines the plasma membrane during interphase and plays a central role in directional cell expansion. Microtubule-associated proteins (MAPs) decorate cortical MTs with distinct patterns, regulating MT dynamic instability, MT severing, and other array-ordering processes. The Arabidopsis root has emerged as a highly useful system for identifying and studying cell-expansion-related MAPs. Here, we review how cortical MTs are thought to behave and become ordered in expanding root cells, and we discuss the emerging picture of how MAPs fundamentally govern MT ordering and directional growth processes.     

Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic–AMP–protein kinase A–dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1–deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.     

Calprotectin: A protein related to cardiovascular risk in adult patients with obstructive sleep apnea

IntroductionIncreased levels of inflammatory mediators, such as hs-CRP, have been detected in patients with obstructive sleep apnea (OSA) and used as cardiovascular risk and disease outcome predictors. Calprotectin is an inflammatory marker regulating atherogenic processes not investigated in adult OSA patients. The aim of the present study as primary objective was to examine the role of calprotectin as an inflammatory molecule, acting through a distinct pathway to the atherogenic process in adult OSA patients and its associations with hs-CRP and the lipidemic profile of the patients. As a secondary objective was the evaluation of the atherogenic markers post-CPAP treatment.Materials and methodsSeventy-four participants underwent full overnight polysomnography. Blood samples were collected for calprotectin, hs-CRP, total cholesterol, triglycerides, LDL, HDL and glucose levels. Thirty-two OSA patients were reexamined 6 months post-CPAP treatment.ResultsOut of 74 participants included in the study, 33 had moderate OSA, 27 had severe OSA and 14 were controls. Calprotectin and hs-CRP were significantly increased in patients with moderate and severe OSA compared to controls (p < 0.0001). Calprotectin and hs-CRP levels were positively correlated with apnea-hypopnea index, BMI and total time of sleep with SaO₂ <90% and inversely correlated with SaO₂ minimum and mean values. Calprotectin and hs-CRP levels were significantly improved post-CPAP treatment (p < 0.0001).DiscussionCalprotectin may serve as a novel and reliable, biomarker of cardiovascular risk severity in OSA patients. The decrease of calprotectin levels post-CPAP treatment combined with hs-CRP amelioration could provide evidence for reduction of cardiovascular risk post CPAP treatment.     

The loss of virulence of histone H1 overexpressing Leishmania donovani parasites is directly associated with a reduction of HSP83 rate of translation

Overexpression of Leishmania histone H1 (LeishH1) was previously found to cause a promastigote‐to‐amastigote differentiation handicap, deregulation of cell‐cycle progression, and loss of parasite infectivity. The aim of this study was to identify changes in the proteome of LeishH1 overexpressing parasites associated with the avirulent phenotype observed. 2D‐gel electrophoresis analysis revealed only a small protein subset of differentially expressed proteins in the LeishH1 overexpressing promastigotes. Among these was the chaperone HSP83, known for its protective role in Leishmania drug‐induced apoptosis, which displayed lower translational rates. To investigate if the lower expression levels of HSP83 are associated with the differentiation handicap, we assayed the thermostability of parasites by subjecting them to heat‐shock (25°C→37°C), a natural stress‐factor occurring during stage differentiation. Heat‐shock promoted apoptosis to a greater extent in the LeishH1 overexpressing parasites. Interestingly, these parasites were not only more sensitive to heat‐shock but also to drug‐induced [Sb(III)] cell‐death. In addition, the restoration of HSP83 levels re‐established drug resistance, and restored infectivity to LeishH1 overexpressing parasites in the murine J774 macrophage model. Overall, this study suggests that LeishH1 levels are critical for the parasite's stress‐induced adaptation within the mammalian host, and highlights the cross‐talk between pathways involved in drug resistance, apoptosis and virulence.     

Blinded by PRISMA: Are Systematic Reviewers Focusing on PRISMA and Ignoring Other Guidelines?

Background

PRISMA guidelines have been developed to improve the reporting of systematic reviews (SRs). Other reporting guidelines and techniques to assess methodological quality of SRs have been developed. We aimed to assess the frequency of the use of reporting and other guidelines in SRs to assess whether PRISMA is being used inappropriately as a substitute for other relevant guidelines.

Methods

Web of Knowledge was searched to identify articles citing the PRISMA guidelines over a 12-month period. The use of reporting guidelines (including PRISMA and MOOSE) and tools for assessing methodological quality (including QUADAS) was assessed. Factors associated with appropriate use of guidelines including review type, field of publication and involvement of a methodologist were investigated.

Results

Over the 12-month period, 701 SRs were identified. MOOSE guidelines were cited in just 17% of epidemiologic reviews; QUADAS or QUADAS-2 was referred to in just 40% of diagnostic SRs. In the multivariable analysis, medical field of publication and methodologist involvement (OR = 1.97, 95% CI: 1.37, 2.83) were significant predictors of appropriate use of guidelines. Inclusion of a meta-analysis resulted in 73% higher odds of appropriate usage of systematic review guidelines (OR = 1.73, 95% CI: 1.22, 2.35).

Conclusions

Usage of SR reporting guidelines and tools for assessment of methodological quality other than PRISMA may be under-utilized with negative implications both for the reporting and methodological quality of systematic reviews.     

Hsp90 canalizes developmental perturbation

Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of 'client' proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.     

Outcome Discrepancies and Selective Reporting: Impacting the Leading Journals?

BackgroundSelective outcome reporting of either interesting or positive research findings is problematic, running the risk of poorly-informed treatment decisions. We aimed to assess the extent of outcome and other discrepancies and possible selective reporting between registry entries and published reports among leading medical journals.MethodsRandomized controlled trials published over a 6-month period from July to December 31^st, 2013, were identified in five high impact medical journals: The Lancet, British Medical Journal, New England Journal of Medicine, Annals of Internal Medicine and Journal of American Medical Association were obtained. Discrepancies between published studies and registry entries were identified and related to factors including registration timing, source of funding and presence of statistically significant results.ResultsOver the 6-month period, 137 RCTs were found. Of these, 18% (n = 25) had discrepancies related to primary outcomes with the primary outcome changed in 15% (n = 20). Moreover, differences relating to non-primary outcomes were found in 64% (n = 87) with both omission of pre-specified non-primary outcomes (39%) and introduction of new non-primary outcomes (44%) common. No relationship between primary or non-primary outcome change and registration timing (prospective or retrospective; P = 0.11), source of funding (P = 0.92) and presence of statistically significant results (P = 0.92) was found.ConclusionsDiscrepancies between registry entries and published articles for primary and non-primary outcomes were common among trials published in leading medical journals. Novel approaches are required to address this problem.