Syndromic Insulin Resistance: Models for the Therapeutic Basis of the Metabolic Syndrome and Other Targets of Insulin Resistance

ObjectiveTo investigate the link between insulin resistance and the metabolic syndrome how to develop treatment approaches.MethodsWe present 3 cases of extreme syndromic insulin resistance: lipodystrophy, autoantibodies to the insulin receptor, and mutations in the insulin receptor gene, with accompanying discussion of pathophysiology and treatment.ResultsIn lipodystrophy, insulin resistance is a direct consequence of leptin deficiency, and thus leptin replacement reverses metabolic syndrome abnormalities, including diabetes and hypertriglyceridemia. The insulin “receptoropathies,” including autoantibodies to the insulin receptor and insulin receptor gene mutations, are characterized by extreme insulin resistance and ovarian hyperandrogenism, without dyslipidemia or fatty liver disease. Autoantibodies to the insulin receptor can be treated using an immunosuppressive paradigm adapted from treatment of other autoimmune and neoplastic conditions. Leptin treatment has shown some success in treating hyperglycemia in patients with insulin receptor gene mutations. Treatment for this condition remains inadequate, and novel therapies that bypass insulin receptor signaling, such as enhancers of brown adipose tissue, are needed.ConclusionsWe present a clinical approach to the treatment of syndromic insulin resistance. The study of rare diseases that replicate the metabolic syndrome, with clear-cut pathophysiology, promotes understanding of novel physiology and development of targeted therapies that may be applicable to the broader population with obesity, insulin resistance, and diabetes. (Endocr Pract. 2012; 18:763-771)     

Epidermal growth factor receptor (EGFR) mutations as biomarker for head and neck squamous cell carcinomas (HNSCC)

Abstract

Context: Mutations in tyrosine kinase domain (TK) of epidermal growth factor receptor (EGFR) lead to signalling interruptions in several cancers.

Objective: To understand EGFR mutations in head and neck squamous cell carcinomas (HNSCC), and their role as biomarkers.

Methods: Screened 129 HNSCC patients and 150 controls for mutations in the TK domain using polymerase chain reaction (PCR), single strand confirmatory polymorphism (SSCP) and sequencing.

Results: 81.39% of HNSCC had four mutations: G2155C, G2176A, C2188G and G2471A among these two mutations were also reported in other cancers where as two novel mutations are being reported for the first time in HNSCC. Mutational frequency was significantly associated with an advanced stage of HNSCC, habits of tobacco/alcohol and ages above 49 years.

Conclusion: EGFR single nucleotide polymorphisms could be useful biomarkers of HNSCC.     

Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain

BackgroundLung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of “acquired” resistance.ConclusionIn patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.     

Mild Androgen Insensitivity Syndrome: The Current Landscape

ObjectiveMild androgen insensitivity syndrome (MAIS) belongs to the androgen insensitivity syndrome (AIS) spectrum, an X-linked genetic disease that is the most common cause of differences in sex development. Unfortunately, AIS studies mainly focus on the partial and complete phenotypes, and the mild phenotype (MAIS) has been barely reported. Our purpose is to explore the MAIS facets, clinical features, and molecular aspects.MethodsWe collected all reported MAIS cases in the medical literature and presented them based on the phenotype and molecular diagnosis.ResultsWe identified 49 different androgen receptor (AR) mutations in 69 individuals in the literature. We compared the AR mutations presented in individuals with MAIS with AR mutations previously reported in other AIS phenotypes (partial and complete) regarding the type, location, genotype-phenotype correlation, and functional studies.ConclusionThis review provides a landscape of the mild phenotype of AIS. Most patients with MAIS present with male factor infertility. Therefore, AR gene sequencing should be considered during male factor infertility investigation, even in males with typically male external genitalia. In addition, MAIS can be part of other medical conditions, such as X-linked spinal and bulbar muscular atrophy (Kennedy disease).     

Minireview: Constitutively Active G Protein-Coupled Receptors: Potential Mechanisms of Receptor Activation

Abstract

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.     

Effect of thermostable mutations on the neurotensin receptor 1 (NTSR1) activation state

Abstract

Neurotensin (NTS) is a 13-amino acid neuropeptide with neuroendocrine and vasoactive functions that is widely expressed in the central nervous system and gastrointestinal tract. NTS is sensed by a multiple cell surface proteins including two G protein-coupling receptors (GPCRs): NTS receptors 1 and 2 (NTSR₁ and NTSR₂). Crystal structures of NTSR₁ have successfully elucidated agonist binding within the orthosteric pocket of receptor but have not revealed the full activation state of the receptor. Recent studies have attempted to address this challenge by improving NTSR₁ crystal formation via thermostable mutants; unfortunately, these mutations exhibit functional defects in the G protein coupling of NTSR₁. Here, we have used molecular dynamics simulations to gain greater insights into how the amino acid substitutions used in these thermostable mutants (E166A, L310A and F358A) impact receptor activation. Our simulations indicate that wild-type NTSR₁ in complex with NTS_8-13 shows more active-like features including a 17.7?Å shift in TM6, reflecting a network of polar and aromatic interactions orchestrating agonist-induced receptor conformational changes. We also provide evidence indicating that F358 is a precursor to the rotamer change observed in W321, and our collective analysis also suggests that mutations E166A and F358A are less impactful to G protein coupling than L310A. Furthermore, we believe that our findings can be used to design future NTSR₁ mutants that do not interfere with agonist-induced conformational changes and downstream G protein coupling and thus produce structures that will allow visualization of the fully activated receptor conformation.     

Non-neutralizing antibodies increase endogenous circulating Ang1 levels

ABSTRACT

Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies’ pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies. Nevertheless, we showed that non-neutralizing antibodies may have therapeutic potential by increasing the level of a target molecule in the circulation.     

AFFINITY LABELING OF CYSTEINE-MUTANTS EVIDENCES CONTACT RESIDUES IN MODELED RECEPTOR BINDING SITES

ABSTRACT

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABA_A receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M₂ segment of the α₁ subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.     

Transcriptional Profile of Muscle following Acute Induction of Symptoms in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy

BackgroundKennedy’s disease/Spinobulbar muscular atrophy (KD/SBMA) is a degenerative neuromuscular disease affecting males. This disease is caused by polyglutamine expansion mutations of the androgen receptor (AR) gene. Although KD/SBMA has been traditionally considered a motor neuron disease, emerging evidence points to a central etiological role of muscle. We previously reported a microarray study of genes differentially expressed in muscle of three genetically unique mouse models of KD/SBMA but were unable to detect those which are androgen-dependent or are associated with onset of symptoms.Conclusions/SignificanceBy comparing the current results with those from the three previously reported models we were able to identify KD/SBMA candidate genes that are androgen dependent, and occur early in the disease process, properties which are promising for targeted therapeutics.     

Molecular characterization of exons 6, 8 and 9 of ABCB4 gene in children with Progressive Familial Intrahepatic Cholestasis type 3

Aim of work: To estimate the frequency of mutations involving exons 6, 8 and 9 of Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene among children with progressive intrahepatic cholestasis with high γ-GT activity (PFIC3).

Subjects and methods: Cross sectional study was conducted on 30 children with PFIC3. Genotyping was performed by sequencing analysis of exons 6, 8 and exon 9 of ABCB4 gene.

Results: Heterozygous synonymous polymorphic variant was detected in exon 6 (rs 1202283) and in exon 8 (rs 2109505). No mutations in studied exons were detected.

Conclusion: Exons 6, 8 and 9 mutations of ABCB4 gene are not common among Egyptian children with PFIC3.     

Transferability of thermostabilizing mutations between β-adrenergic receptors

Abstract

In previous work we described six point mutations that thermostabilised the turkey β₁-adrenergic receptor (tβ₁AR). The thermostable mutant, tβ₁AR-m23, had an apparent T_m 21°C higher than the native protein when solubilized in dodecylmaltoside (DDM) and, in addition, was significantly more stable in short chain detergents, which allowed its crystallization and structure determination. Identification of thermostabilizing mutations in tβ₁AR was performed by systematic mutagenesis followed by expressing and assaying each of the 318 mutants for their thermostability. This is time-consuming, so to facilitate studies on related receptors, we have studied the transferability of these mutations to the human adrenergic receptors, hβ₁AR and hβ₂AR, which have, respectively, 76% and 59% sequence identity to tβ₂AR, excluding the N- and C-termini. Thermostability assays revealed that hβ₁AR was much more unstable than tβ₂AR, whereas hβ₂AR was more stable than tβ₁AR. Addition of the 6 thermostabilizing mutations in tβ₂AR-m23 into both hβ₂AR and hβ₂AR increased their apparent T_ms by 17°C and 11°C, respectively. In addition, the mutations affected the global conformation of the human receptors so that they were predominantly in the antagonist bound form, as was originally observed for tβ₂AR-m23. Thus, once thermostabilizing mutations have been identified in one G protein-coupled receptor, stabilization of close members within the subfamily is rapidly obtainable.     

SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力

【目的】探究铜绿假单胞菌(Pseudomonas aeruginosa)鸟苷酸环化酶(diguanylate cyclase,DGC)SadC合成的环二鸟苷酸(cyclicdi-GMP,c-di-GMP)信号与PilZ结构域受体间的信号传递关系,分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析;SadC过表达背景下,PilZ结构域受体突变各菌株的泳动表型分析和筛选;基因敲除和过表达解析筛选出的PilZ结构域受体功能;定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力;PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制;功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强,而过表达PilZ或FlgZ则抑制了泳动能力;定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要,多序列比对分析表明这些位点是其保...     

Molecular Docking and Interaction Studies of Identified Abscisic Acid Receptors in Oryza sativa: An In-Silico Perspective on Comprehending Stress Tolerance Mechanisms

BackgroundAbiotic stresses affect plants in several ways and as such, phytohormones such as abscisic acid (ABA) play an important role in conferring tolerance towards these stresses. Hence, to comprehend the role of ABA and its interaction with receptors of the plants, a thorough investigation is essential.AimThe current study aimed to identify the ABA receptors in Oryza sativa, to find the receptor that binds best with ABA and to examine the mutations present to help predict better binding of the receptors with ABA.MethodsProtein sequences of twelve PYL (Pyrabactin resistance 1) and seven PP2C (type 2C protein phosphatase) receptors were retrieved from the Rice Annotation Project database and their 3D structures were predicted using RaptorX. Protein-ligand molecular docking studies between PYL and ABA were performed using AutoDock 1.5.6, followed by 100ns molecular dynamic simulation studies using Desmond to determine the acceptable conformational changes after docking via root mean square deviation RMSD plot analysis. Protein-protein docking was then carried out in three sets: PYL-PP2Cs, PYL-ABA-PP2C and PYL(mut)-ABA-PP2C to scrutinize changes in structural conformations and binding energies between complexes. The amino acids of interest were mapped at their respective genomic coordinates using SNP-seek database to ascertain if there were any naturally occurring single nucleotide polymorphisms (SNPs) responsible for triggering rice PYLs mutations.ResultsInitial protein-ligand docking studies revealed good binding between the complexes, wherein PYL6-ABA complex showed the best energy of -8.15 kcal/mol. The 100ns simulation studies revealed changes in the RMSD values after docking, indicating acceptable conformational changes. Furthermore, mutagenesis study performed at specific PYL-ABA interacting residues followed by downstream PYL(mut)-ABA-PP2C protein-protein docking results after induction of mutations demonstrated binding energy of -8.17 kcal/mol for PP2C79-PYL11-ABA complex. No naturally occurring SNPs that were responsible for triggering rice PYL mutations were identified when specific amino acid coordinates were mapped at respective genomic coordinates.ConclusionThus, the present study provides valuable insights on the interactions of ABA receptors in rice and induced mutations in PYL11 that can enhance the downstream interaction with PP2C.     

Multitasking discoidin domain receptors are involved in several and specific hallmarks of cancer

ABSTRACT

Discoidin domain receptors, DDR1 and DDR2, are two members of collagen receptor family that belong to tyrosine kinase receptor subgroup. Unlike other matrix receptor-like integrins, these collagen receptors have not been extensively studied. However, more and more studies are focusing on their involvement in cancer. These two receptors are present in several subcellular localizations such as intercellular junction or along type I collagen fibers. Consequently, they are involved in multiple cellular functions, for instance, cell cohesion, proliferation, adhesion, migration and invasion. Furthermore, various signaling pathways are associated with these multiple functions. In this review, we highlight and characterize hallmarks of cancer in which DDRs play crucial roles. We discuss recent data from studies that demonstrate the involvement of DDRs in tumor proliferation, cancer mutations, drug resistance, inflammation, neo-angiogenesis and metastasis. DDRs could be potential targets in cancer and we conclude this review by discussing the different ways to inhibits them.     

Genotype-Phenotype Correlations in CYP1B1-Associated Primary Congenital Glaucoma Patients Representing Two Large Cohorts from India and Brazil

BackgroundPrimary congenital glaucoma (PCG), occurs due to the developmental defects in the trabecular meshwork and anterior chamber angle in children. PCG exhibits genetic heterogeneity and the CYP1B1 gene has been widely implicated worldwide. Despite the diverse mutation spectra, the clinical implications of these mutations are yet unclear. The present study attempted to delineate the clinical profile of PCG in the background of CYP1B1 mutations from a large cohort of 901 subjects from India (n=601) and Brazil (n=300).MethodsGenotype-phenotype correlations was undertaken on clinically well characterized PCG cases from India (n=301) and Brazil (n=150) to assess the contributions of CYP1B1 mutation on a set of demographic and clinical parameters. The demographic (gender, and history of consanguinity) and quantitative clinical (presenting intraocular pressure [IOP] and corneal diameter [CD]) parameters were considered as binary and continuous variables, respectively, for PCG patients in the background of the overall mutation spectra and also with respect to the prevalent mutations in India (R368H) and Brazil (4340delG). All these variables were fitted in a multivariate logistic regression model using the Akaike Information Criterion (AIC) to estimate the adjusted odds ratio (OR) using the R software (version 2.14.1).ResultsThe overall mutation spectrum were similar across the Indian and Brazilian PCG cases, despite significantly higher number of homozygous mutations in the former (p=0.024) and compound heterozygous mutations in the later (p=0.012). A wide allelic heterogeneity was observed and only 6 mutations were infrequently shared between these two populations. The adjusted ORs for the binary (demographic) and continuous (clinical) variables did not indicate any susceptibility to the observed mutations (p>0.05).ConclusionsThe present study demonstrated a lack of genotype-phenotype correlation of the demographic and clinical traits to CYP1B1 mutations in PCG at presentation. However, the susceptibility of these mutations to the long-term progression of these traits are yet to be deciphered.     

Recombinant Human Parathyroid Hormone Therapy (1-34) In an Adult Patient with a Gain-of-Function Mutation in the Calcium-Sensing Receptor-a Case Report

ObjectiveTo describe a case of hypocalcemia in a patient with a gain-of-function mutation in the calcium-sensing receptor that was undetected until adulthood and successfully treated with recombinant parathyroid hormone.MethodsThe clinical findings, laboratory data, and a review of the pertinent literature are presented.ResultsA 55-year-old woman was hospitalized and seen by the endocrinology consult service for hypocalcemia that was refractory to repeated doses of intravenous calcium gluconate. She expressed concern about chronic leg muscle cramps and paresthesias of the lips and fingertips. In addition, she had no history of neck surgery, neck irradiation, or any autoimmune disease. She was a well-appearing female with no dysmorphic features or skin changes. Laboratory tests revealed hypocalcemia, hyperphosphatemia, hypomagnesemia, and hypovitamino-sis D. Her parathyroid hormone concentration (PTH) was low at 14.2 pg/mL. Her PTH and calcium concentrations remained low despite repletion of magnesium and treatment with calcitriol and oral calcium replacement. A 24-hour collection for urinary calcium showed inappropriate hypercalciuria. Medical records showed her hypocalcemia to be chronic. Additionally, several family members had also complained of muscle cramps. A congenital cause of her hypoparathyroidism was considered, and genetic testing confirmed heterozygosity for a gain-of-function mutation in the calcium-sensing receptor gene associated with autosomal dominant familial isolated hypoparathyroidism (ADH). Treatment with subcutaneous recombinant human parathyroid hormone teriparatide (rhPTH [1-34]) 20 mcg twice daily for three days normalized her calcium and phosphorus concentrations.ConclusionrhPTH (1-34) is an effective treatment for patients with hypoparathyroidism due to gain-of-function mutations in the calcium-sensing receptor. ADH can be insidious in presentation and the diagnosis can be missed unless there is a high index of suspicion. (Endocr Pract. 2013;19:e24-e28)     

Effect of Novel Amino Acids and Dipeptides Substituted 3-Morpholino Arecoline Derivatives as Muscarinic Receptor 1 Agonists in Alzheimer’s Dementia Models

A series of novel, potent and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester group of arecoline (which is reported as mucarinic agonist) has been replaced by N-substituted morpholine ring. The structure activity relationship reveals that the increase in lipophilic carbon chain on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor. In the present study, we are reporting N-amino acid substituted 9(a–k) and dipeptides substituted 10(a–j) and 11(a–j) morpholino arecoline derivatives, along with their in vitro muscarinic binding studies by using [³H]QNB and also in vivo evaluation of memory and learning in male Wistar rats (passive avoidance test plus maze studies) as M1 receptor agonist. Some molecules from the dipeptide series (10b, 10c and 10j) showed potent M1 receptor agonist activity. Other derivatives also showed considerable M1 receptor binding affinity.