Tatiana V. Novoselova

Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth. Disruption of a protein required for effective signaling by a melanocortin receptor causes severe obesity in mice. Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

Treatment with extracellular HSP70/HSC70 protein can reduce polyglutamine toxicity and aggregation

The accumulation of insoluble protein aggregates is a feature of neurodegenerative disease. Overexpression of Heat Shock Protein 70 (HSP70) can protect cells with protein aggregates from apoptosis. Another trait of HSP70 is its ability to cross the plasma membrane. Therefore, we purified a preparation of HSP70/HSC70 from bovine muscle and used it in a model of Huntingtons disease. Human neuroblastoma SK‐N‐SH cells were transfected with huntington exon 1 with short (25) or long (103) CAG trinucleotide repeats coupled to green flourescent protein (GFP). Cells transfected with the long polyCAG repeat had insoluble protein aggregates and died through apoptosis. Biotinylated HSP70/HSC70 incorporated into the culture medium appeared inside the cells within 3–6u2003h of incubation. This incorporation correlated with a reduction in apoptotic cells by 40–50%. Confocal microscopy revealed that labelled internalized HSP70/HSC70 co‐localized with the polyglutamine inclusions. The measurement of the number and size of inclusions showed that HSP70/HSC70 was able to reduce both these parameters. A filter trap assay and immunoblotting demonstrated that the introduction of HSP70/HSC70 also decreased protein aggregation. Together with earlier data on the effects of exogenously administered HSP70/HSC70 on cultured cells and on animals, these data show that preparations based on HSP70 may have some potential as therapies for a variety of neurodegenerative pathologies.

The X-linked retinitis pigmentosa protein RP2 facilitates G protein traffic

The X-linked retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins are implicated in the traffic of proteins to the primary cilia. Here, we show that RP2 can facilitate the traffic of the Gβ subunit of transducin (Gβ1). Glutathione S-transferase (GST)-RP2 pulled down Gβ from retinal lysates and the interaction was specific to Gβ1, as Gβ3 or Gβ5L did not bind RP2. RP2 did not appear to interact with the Gβ:Gγ heterodimer, in contrast Gγ1 competed with RP2 for Gβ binding. Overexpression of Gβ1 in SK-N-SH cells led to a cytoplasmic accumulation of Gβ1, while co-expression of RP2 or Gγ1 with Gβ1 restored membrane association of Gβ1. Furthermore, RP2 small interfering RNA in ARPE19 cells resulted in a reduction in Gβ1 membrane association that was rescued by Gγ1 overexpression. The interaction of RP2 with Gβ1 required RP2 N-terminal myristolyation and the co-factor C (TBCC) homology domain. The interaction was also disrupted by the pathogenic mutation R118H, which blocks Arl3 GAP activity. Interestingly, Arl3-Q71L competed with Gβ1 for RP2 binding, suggesting that Arl3-GTP binding by RP2 would release Gβ1. RP2 also stimulated the association of Gβ1 with Rab11 vesicles. Collectively, the data support a role for RP2 in facilitating the membrane association and traffic of Gβ1, potentially prior to the formation of the obligate Gβ:Gγ heterodimer. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in the cilia-associated traffic of G proteins.

Melanocortin receptor accessory proteins in adrenal gland physiology and beyond

The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R-MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic-pituitary-adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis. In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.

Whole-Exome Sequencing in the Differential Diagnosis of Primary Adrenal Insufficiency in Children.

Adrenal insufficiency is a rare, but potentially fatal medical condition. In children, the cause is most commonly congenital and in recent years a growing number of causative gene mutations have been identified resulting in a myriad of syndromes that share adrenal insufficiency as one of the main characteristics. The evolution of adrenal insufficiency is dependent on the variant and the particular gene affected, meaning that rapid and accurate diagnosis is imperative for effective treatment of the patient. Common practice is for candidate genes to be sequenced individually, which is a time-consuming process and complicated by overlapping clinical phenotypes. However, with the availability, and increasing cost effectiveness of whole-exome sequencing, there is the potential for this to become a powerful diagnostic tool. Here, we report the results of whole-exome sequencing of 43 patients referred to us with a diagnosis of familial glucocorticoid deficiency (FGD) who were mutation negative for MC2R, MRAP, and STAR the most commonly mutated genes in FGD. WES provided a rapid genetic diagnosis in 17/43 sequenced patients, for the remaining 60% the gene defect may be within intronic/regulatory regions not covered by WES or may be in gene(s) representing novel etiologies. The diagnosis of isolated or familial glucocorticoid deficiency was only confirmed in 3 of the 17 patients, other genetic diagnoses were adrenal hypo- and hyperplasia, Triple A, and autoimmune polyendocrinopathy syndrome type I, emphasizing both the difficulty of phenotypically distinguishing between disorders of PAI and the utility of WES as a tool to achieve this.

Production of Heat Shock Proteins, Cytokines, and Nitric Oxide in Toxic Stress

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-α, TNF-β), interferon-γ (IFN-γ), interleukin-2,-3,-6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-α in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-α, TNF-β, and NO in mouse cells. The production of other cytokines (interleukins and IFN-γ) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

ABSTRACT Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.

Central role for melanocortin-4 receptors in offspring hypertension arising from maternal obesity

Significance Obesity is increasing in pregnant women worldwide. Independent associations have been reported between maternal obesity and metabolic cardiorenal disorders in the offspring, including hypertension. In this study, using genetically modified mice, we have identified a role for the hypothalamic paraventricular nucleus (PVH) melanocortin system in the etiology of hypertension. We show that maternal obesity permanently resets the responsiveness of the central sympathetic nervous system via this pathway. We conclude that neonatal leptin exposure is the primary mediator, because exogenous neonatal leptin administration to pups of lean mice leads to the same phenotype mediated by PVH melanocortin-4 receptors. Thus, primary hypertension of sympathetic origin can result from early-life exposure to maternal obesity, and the melanocortin pathway presents a target for intervention. Melanocortin-4 receptor (Mc4r)–expressing neurons in the autonomic nervous system, particularly in the paraventricular nucleus of the hypothalamus (PVH), play an essential role in blood pressure (BP) control. Mc4r-deficient (Mc4rKO) mice are severely obese but lack obesity-related hypertension; they also show a reduced pressor response to salt loading. We have previously reported that lean juvenile offspring born to diet-induced obese rats (OffOb) exhibit sympathetic-mediated hypertension, and we proposed a role for postnatally raised leptin in its etiology. Here, we test the hypothesis that neonatal hyperleptinemia due to maternal obesity induces persistent changes in the central melanocortin system, thereby contributing to offspring hypertension. Working on the OffOb paradigm in both sexes and using transgenic technology to restore Mc4r in the PVH of Mc4rKO (Mc4rPVH) mice, we have now shown that these mice develop higher BP than Mc4rKO or WT mice. We have also found that experimental hyperleptinemia induced in the neonatal period in Mc4rPVH and WT mice, but not in the Mc4rKO mice, leads to heightened BP and severe renal dysfunction. Thus, Mc4r in the PVH appears to be required for early-life programming of hypertension arising from either maternal obesity or neonatal hyperleptinemia. Early-life exposure of the PVH to maternal obesity through postnatal elevation of leptin may have long-term consequences for cardiovascular health.

Loss of Mrap2 is associated with Sim1 deficiency and increased circulating cholesterol

Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2tm1a/tm1a) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.

NNT Pseudoexon Activation as a Novel Mechanism for Disease in Two Siblings With Familial Glucocorticoid Deficiency

Context: Intronic DNA frequently encodes potential exonic sequences called pseudoexons. In recent years, mutations resulting in aberrant pseudoexon inclusion have been increasingly recognized to cause disease. Objectives: To find the genetic cause of familial glucocorticoid deficiency (FGD) in two siblings. Patients: The proband and his affected sibling, from nonconsanguineous parents of East Asian and South African origin, were diagnosed with FGD at the ages of 21 and 8 months, respectively. Design: Whole exome sequencing was performed on genomic DNA (gDNA) of the siblings. Variants in genes known to cause FGD were assessed for causality. Further analysis of gDNA and cDNA was performed by PCR/RT-PCR followed by automated Sanger sequencing. Results: Whole exome sequencing identified a single, novel heterozygous variant (p.Arg71*) in nicotinamide nucleotide transhydrogenase (NNT) in both affected individuals. Follow-up cDNA analysis in the proband identified a 69-bp pseudoexon inclusion event, and Sanger sequencing of his gDNA identified a 4-bp duplication responsible for its activation. The variants segregated with the disease: p.Arg71* was inherited from the mother, the pseudoexon change was inherited from the father, and an unaffected sibling had inherited only the p.Arg71* variant. Conclusions: FGD in these siblings is caused by compound heterozygous mutations in NNT; one causing pseudoexon inclusion in combination with another leading to Arg71*. Discovery of this pseudoexon activation mutation highlights the importance of identifying sequence changes in introns by cDNA analysis. The clinical implications of these findings include: facilitation of antenatal genetic diagnosis, early institution of potentially lifesaving therapy, and the possibility of preventative or curative intervention.