Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

Familial multiple sclerosis in patients with Von Hippel-Lindau disease.

BACKGROUND: Multiple sclerosis (MS) is a progressive autoimmune demyelinating disorder. Recent studies suggest that a combination of genetic susceptibility and environmental insult contributes to its pathogenesis. Many candidate genes have been discovered to modulate susceptibility for developing MS by genome wide association studies (GWAS); these include major histocompatibility complex (MHC) genes and non-MHC genes. MS cases in the context of genetic diseases may provide different approaches and clues towards identifying novel genes and pathways involved in MS pathogenesis. Here, we present a case series of two related patients with concomitant Von Hippel-Lindau disease (VHLD) and MS. CASE PRESENTATION: We present two patients, a mother (case 1) and daughter (case 2), who developed superimposed relapsing-remitting multiple sclerosis in the background of the autosomal dominant genetic disorder VHLD. Several tumors characteristic of VHLD developed in both cases with pancreatic and renal neoplasms and cerebellar hemangioblastomas. In addition, both patients developed clinical symptoms consistent with multiple sclerosis, supported by radiologic lesions disseminating in time and space. CONCLUSION: Though non-MHC susceptibility genes remain elusive in MS, we present the striking finding of superimposed multiple sclerosis in a mother and daughter with VHLD. The VHL gene is known to be the primary regulator of Nrf2, the well-established target of the FDA-approved therapeutic dimethyl fumarate. These cases provide support for further studies to determine whether VHLD pathway related genes represent a novel genetic link in multiple sclerosis.

Monomeric human soluble CD4 dimerizes at physiological temperature: VTSAXS data based modeling and screening of retardant molecules.

Earlier, solution small angle X-ray scattering (SAXS) data at 10 °C showed that soluble CD4 (sCD4; 1 mg/ml) is monomer with shape similar to single chain in crystal structures of its dimer. Query remained whether the dimeric state of CD4 can form independent of packing effects of crystal? Taking cue from other systems, we explored heat induced possible association of native shapes of sCD4 by variable temperature SAXS (VTSAXS) experiments. The predominant particle size increased consistently with temperature and around 35-40 °C, the estimated mass indicated dimeric state in solution. Furthermore, the observed association was found to be reversible to certain extent. Using SAXS profile representing dimer and crystal structure of monomer, we solved models of CD4 dimers which were dominated by D4-D4 interactions and appeared "wobbling" about the crystal structure of dimeric CD4, convincing pre-existence of crystal-like association in solution. To break the dimerization, we theoretically screened for small molecules binding to dimeric interface of D4 domain. Additionally, as negative control or not expecting to interfere, we searched molecules preferentially docking on the apex of D1 domain. VTSAXS experiments of CD4 + molecules at ∼1:3 molar ratio showed that as expected few D4 reactive hits could retard dimerization, yet surprisingly molecules which docked at D1 domain could also derail dimerization. Additional analysis led to conclusion that there lies a systematic communication network across the structural length of sCD4 which senses binding to self and other molecules, and our work can be used to develop new (or re-purpose known) molecules as CD4-reactive immunosuppressive agents.Communicated by Ramaswamy H. Sarma.

Heat induces end to end repetitive association in P. furiosus L-asparaginase which enables its thermophilic property.

It remains undeciphered how thermophilic enzymes display enhanced stability at elevated temperatures. Taking L-asparaginase from P. furiosus (PfA) as an example, we combined scattering shapes deduced from small-angle X-ray scattering (SAXS) data at increased temperatures with symmetry mates from crystallographic structures to find that heating caused end-to-end association. The small contact point of self-binding appeared to be enabled by a terminal short ß-strand in N-terminal domain, Leu179-Val-Val-Asn182 (LVVN). Interestingly, deletion of this strand led to a defunct enzyme, whereas suplementation of the peptide LVVN to the defunct enzyme restored structural frameworkwith mesophile-type functionality. Crystal structure of the peptide-bound defunct enzyme showed that one peptide ispresent in the same coordinates as in original enzyme, explaining gain-of lost function. A second peptide was seen bound to the protein at a different location suggesting its possible role in substrate-free molecular-association. Overall, we show that the heating induced self-assembly of native shapes of PfA led to an apparent super-stable assembly.

MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease.

Polyglutamine (polyQ) tract expansion leads to proteotoxic misfolding and drives a family of nine diseases. We study spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder of the neuromuscular system caused by the polyQ androgen receptor (AR). Using a knock-in mouse model of SBMA, AR113Q mice, we show that E3 ubiquitin ligases which are a hallmark of the canonical muscle atrophy machinery are not induced in AR113Q muscle. Similarly, we find no evidence to suggest dysfunction of signaling pathways that trigger muscle hypertrophy or impairment of the muscle stem cell niche. Instead, we find that skeletal muscle atrophy is characterized by diminished function of the transcriptional regulator Myocyte Enhancer Factor 2 (MEF2), a regulator of myofiber homeostasis. Decreased expression of MEF2 target genes is age- and glutamine tract length-dependent, occurs due to polyQ AR proteotoxicity, and is associated with sequestration of MEF2 into intranuclear inclusions in muscle. Skeletal muscle from R6/2 mice, a model of Huntington disease which develops progressive atrophy, also sequesters MEF2 into inclusions and displays age-dependent loss of MEF2 target genes. Similarly, SBMA patient muscle shows loss of MEF2 target gene expression, and restoring MEF2 activity in AR113Q muscle rescues fiber size and MEF2-regulated gene expression. This work establishes MEF2 impairment as a novel mechanism of skeletal muscle atrophy downstream of toxic polyglutamine proteins and as a therapeutic target for muscle atrophy in these disorders.

Author Correction: Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.

Disease Affects Bdnf Expression in Synaptic and Extrasynaptic Regions of Skeletal Muscle of Three SBMA Mouse Models.

Spinal bulbar muscular atrophy (SBMA) is a slowly progressive, androgen-dependent neuromuscular disease in men that is characterized by both muscle and synaptic dysfunction. Because gene expression in muscle is heterogeneous, with synaptic myonuclei expressing genes that regulate synaptic function and extrasynaptic myonuclei expressing genes to regulate contractile function, we used quantitative PCR to compare gene expression in these two domains of muscle from three different mouse models of SBMA: the "97Q" model that ubiquitously expresses mutant human androgen receptor (AR), the 113Q knock-in (KI) model that expresses humanized mouse AR with an expanded glutamine tract, and the "myogenic" model that overexpresses wild-type rat AR only in skeletal muscle. We were particularly interested in neurotrophic factors because of their role in maintaining neuromuscular function via effects on both muscle and synaptic function, and their implicated role in SBMA. We confirmed previous reports of the enriched expression of select genes (e.g., the acetylcholine receptor) in the synaptic region of muscle, and are the first to report the synaptic enrichment of others (e.g., glial cell line-derived neurotrophic factor). Interestingly, all three models displayed comparably dysregulated expression of most genes examined in both the synaptic and extrasynaptic domains of muscle, with only modest differences between regions and models. These findings of comprehensive gene dysregulation in muscle support the emerging view that skeletal muscle may be a prime therapeutic target for restoring function of both muscles and motoneurons in SBMA.

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-sequencing (RNA-Seq) to identify pathways that are disrupted in diseased muscle using AR113Q knockin mice. This analysis unexpectedly identified substantially diminished expression of numerous ubiquitin/proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age, hormone, and glutamine length dependent and arose due to a toxic gain of function conferred by the mutation. Moreover, altered gene expression was associated with decreased levels of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.

The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease.

Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology.

Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8.

Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications.

Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1.

The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane.

Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven their potential as an efficient delivery system due to their intrinsic ability to traverse biological membranes and transport various cargoes into the cells. In the present study, we have identified novel natural protein-derived CPPs using an integrated (in silico and experimental) approach. First, using bioinformatics approach, arginine-rich peptide segments were extracted from SwissProt proteins and their cell-penetrating properties were predicted. Finally, eight peptides were selected and their internalization was validated using experimental techniques. Laser scanning confocal microscopy and flow cytometry confirmed that seven out of eight peptides were internalized into live cells with varying efficiencies without significant cytotoxicity. Three peptides have shown higher internalization efficacy than TAT peptide, the most widely used CPP. Among these three peptides, one peptide (P8), derived from voltage-dependent L-type calcium channel subunit alpha-1D, was able to accumulate inside in a variety of cell types very efficiently through a rapid dose-dependent process. Further, experiments involving inhibition with various endocytic inhibitors along with co-localization studies indicate that the uptake mechanism of P8 is macropinocytosis, a fluid-phase endocytosis process. In addition, competitive inhibition with heparin revealed the involvement of cell-surface proteoglycans in P8 uptake. In summary, the present study provides evidence that an integrated in silico and experimental approach is an effective strategy for the identification of novel CPPs and CPPs identified in the present study have promising perspectives for future drug delivery applications.

Global shape and ligand binding efficiency of the HIV-1-neutralizing antibodies differ from those of antibodies that cannot neutralize HIV-1.

Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. We compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.

Low pH overrides the need of calcium ions for the shape-function relationship of calmodulin: resolving prevailing debates.

Calmodulin (CaM) regulates numerous cellular functions by sensing Ca(2+) levels inside cells. Although its structure as a function of the Ca(2+)-bound state remains hotly debated, no report is available on how pH independently or in interaction with Ca(2+) ions regulates shape and function of CaM. From SAXS data analysis of CaM at different levels of Ca(2+)-ion concentration and buffer pH, we found that (1) CaM molecules possess a Gaussian-chain-like shape in solution even in the presence of Ca(2+) ion or at low pH, (2) the global shape of apo CaM is very similar to its NMR structure rather than the crystal structures, (3) about 16 Ca(2+) ions or more are required per CaM molecule in solution to achieve the four-Ca(2+)-bound crystal structure, (4) low pH alone can impart shape changes in CaM similar to Ca(2+) ions, and (5) at different [Ca(2+)]/[CaM] ratio or pH values, the predominant shape of CaM is essentially a weighted average of its apo and fully activated shape. Results were further substantiated by analysis of sedimentation coefficient values from analytical ultracentrifugation and peptide binding assays using two peptides, each known to preferentially bind the apo or the Ca(2+)-activated state.

Towards strain-independent anti-influenza peptides: a SAXS- and modeling-based study.

Using small angle X-ray scattering (SAXS) data, we reconstructed the scattering shape of the Hemagglutinin (HA) trimer protein from five different influenza strains. Comparison with the known crystal structures-based information aided in identifying volumes pertaining to the glycosylation in the HA trimers. By merging sequence information on HA proteins from pathogenic strains of influenza, we identified a novel druggable pocket composed of residues which remained conserved during evolution, lack propensity to be glycosylated, and play important role in maintaining interchain contacts in the pH-sensitive head group. To test our hypothesis that molecules reactive to this site may retard pH-induced opening of HA trimer in strain-independent manner, we performed in vitro screening of peptides representing interacting epitopes for their ability to retard pH-induced opening of HA trimers. Results brought forth that some of the 20 peptides tested can retard low pH-induced opening/association of HA proteins across different subtypes, thus propagating notion that the drug site and peptides identified here may pave way towards strain-independent anti-influenza molecules.

Catechins protect neurons against mitochondrial toxins and HIV proteins via activation of the BDNF pathway.

Currently, there is no effective treatment for neurological complications of infection with the human immunodeficiency virus that persists despite the use of combination antiretroviral therapy. A medium throughput assay was developed for screening neuroprotective compounds using primary mixed neuronal cells and mitochondrial toxin 3-nitropropionic acid. Using this assay, a library of 2,000 compounds was screened. Out of 256 compounds that showed variable degrees of neuroprotection, nine were related to epicatechin, a monomeric flavonoid found in cocoa and green tea leaves that readily crosses the blood-brain barrier. Hence, catechin, epicatechin, and the related compound, epigallocatechin gallate (EGCG) were further screened for their neuroprotective properties against HIV proteins Tat and gp120, and compared to those of resveratrol. Epicatechin and EGCG targets the brain-derived neurotrophic factor (BDNF) and its precursor proBDNF signaling pathways, normalizing both Tat-mediated increases in proapoptotic proBDNF and concomitant Tat-mediated decreases in the mature BDNF protein in hippocampal neurons. Epicatechin and epigallocatechin gallate were more potent than catechin or resveratrol as neuroprotectants. Due to its simpler structure and more efficient blood-brain barrier penetration properties, epicatechin might be the best therapeutic candidate for neurodegenerative diseases including HIV-associated neurocognitive disorders where oxidative stress is an important pathophysiological mechanism.

Internalized HIV/AIDS-related stigma in a sample of HIV-positive people in Bangladesh.

Internalized stigma among people living with HIV/AIDS (PLHA) is prevalent in Bangladesh. A better understanding of the effects of stigma on PLHA is required to reduce this and to minimize its harmful effects. This study employed a quantitative approach by conducting a survey with an aim to know the prevalence of internalized stigma and to identify the factors associated with internalized stigma among a sample of 238 PLHA (male=152 and female=86) in Bangladesh. The findings suggest that there is a significant difference between groups with the low- and the high-internalized HIV/AIDS stigma in terms of both age and gender. The prevalence of internalized stigma varied according to the poverty status of PLHA. An exploratory factor analysis (EFA) found 10 of 15 items loaded highly on the three factors labelled self-acceptance, self-exclusion, and social withdrawal. About 68% of the PLHA felt ashamed, and 54% felt guilty because of their HIV status. More than half (87.5% male and 19.8% female) of the PLHA blamed themselves for their HIV status while many of them (38.2% male and 8.1% female) felt that they should be punished. The male PLHA more frequently chose to withdraw themselves from family and social gatherings compared to the female PLHA. They also experienced a higher level of internalized stigma compared to the female PLHA. The results suggest that the prevalence of internalized stigma is high in Bangladesh, and much needs to be done by different organizations working for and with the PLHA to reduce internalized stigma among this vulnerable group.

Tropical sprue.

Tropical sprue (TS) is a clinical entity of unknown etiology characterized by an acquired chronic diarrheal illness and malabsorption that affects indigenous inhabitants and expatriates, either long-term residents or short-term visitors, in the tropical countries. The exact pathogenetic sequence of TS remains incompletely characterized. Bacterial overgrowth, disturbed gut motility, and hormonal and histopathologic abnormalities contribute to the development of TS in a susceptible host. Treatment with tetracycline and folate is effective in some patients, although relapses after treatment are common. Research in the areas of microbial factors, pathogenesis, immunogenetics, and hormonal and immune regulation, using modern diagnostic techniques, may be able to settle some of the unanswered issues and open new venues for diagnosis, prevention, and treatment of tropical sprue.

Capsule endoscopy.

Video capsule endoscopy is a major innovation that provides high-resolution imaging of the entire small intestine in its entirety. In the 4 years since its introduction, capsule endoscopy has demonstrated its viability as a first-line investigation in patients with obscure gastrointestinal bleeding after a negative esophagogastroduodenoscopy and colonoscopy, and it has a positive impact on the outcome. Video capsule endoscopy is also useful in the evaluation of inflammatory and neoplastic disorders of the small bowel. Its role in children and in the evaluation of esophageal disorders is currently being explored and defined.