Functional Characterization of Spinocerebellar Ataxia Associated Dynorphin A Mutant Peptides

Mutations in the prodynorphin gene (PDYN) are associated with the development of spinocerebellar ataxia type 23 (SCA23). Pathogenic missense mutations are localized predominantly in the PDYN region coding for the dynorphin A (DynA) neuropeptide and lead to persistently elevated mutant peptide levels with neurotoxic properties. The main DynA target in the central nervous system is the kappa opioid receptor (KOR), a member of the G-protein coupled receptor family, which can elicit signaling cascades mediated by G-protein dissociation as well as β-arrestin recruitment. To date, a thorough analysis of the functional profile for the pathogenic SCA23 DynA mutants at KOR is still missing. To elucidate the role of DynA mutants, we used a combination of assays to investigate the differential activation of G-protein subunits and β-arrestin. In addition, we applied molecular modelling techniques to provide a rationale for the underlying mechanism. Our results demonstrate that DynA mutations, associated with a severe ataxic phenotype, decrease potency of KOR activation, both for G-protein dissociation as well as β-arrestin recruitment. Molecular modelling suggests that this loss of function is due to disruption of critical interactions between DynA and the receptor. In conclusion, this study advances our understanding of KOR signal transduction upon DynA wild type or mutant peptide binding.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5′ and 3′ ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

Acute Onset Flaccid Paralysis as Presentation of Combined Central and Peripheral Demyelination in a Child: A Case Report

Introduction: Inflammatory demyelinating disease like combined central and peripheral demyelination (CCPD) could have varied clinical presentation depending upon the topographical distribution of neural involvement. Case presentation: A seven-year-old child had presented with fever followed by acute onset flaccid paralysis and urinary retention. Weakness in the lower limbs as reported was ascending and symmetric in nature, while no history of trauma, band-like sensation or altered sensorium were documented. Superficial and deep tendon reflexes of both the lower limbs were absent. Routine blood investigations had revealed neutrophilic leucocytosis only. Serum IgM antibody for scrub typhus was found positive. CSF study didn’t show cyto-protein dissociation. NCV had demonstrated absence of F wave and H reflex in the peripheral nerves of lower limbs. Anti-ganglioside antibody profiles were negative. Subsequent investigations including MRI brain and spinal cord had revealed acute onset CCPD. Conclusion: Acute onset combined central and peripheral demyelination in a child had presented as acute flaccid paralysis of the lower limbs and the condition was temporally association with scrub typhus.

Heterogeneity of G-protein activation by the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G-protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messenger proteins or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G-protein activation directly, we took advantage of a recently described NanoBiT G-protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G-proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G-proteins were similarly expressed, suggesting CaSR may activate multiple G-proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.

Probing protein dissociation from gold nanoparticles and the influence of temperature from the protein corona formation mechanism

A protein corona changes protein's structure and characteristics, hindering their identification in situ. Dissociation is an important solution to identify their composition.

Structural insights into differences in G protein activation by family A and family B GPCRs

Family B heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo–electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

Chromosome-autonomous feedback downregulates meiotic DSB competence upon synaptonemal complex formation

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog-engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.

Dynamical control by water at a molecular level in protein dimer association and dissociation

Water, often termed as the “lubricant of life,” is expected to play an active role in navigating protein dissociation–association reactions. In order to unearth the molecular details, we first compute the free-energy surface (FES) of insulin dimer dissociation employing metadynamics simulation, and then carry out analyses of insulin dimerization and dissociation using atomistic molecular-dynamics simulation in explicit water. We select two sets of initial configurations from 1) the dissociated state and 2) the transition state, and follow time evolution using several long trajectories (∼1–2 μs). During the process we not only monitor configuration of protein monomers, but also the properties of water. Although the equilibrium structural properties of water between the two monomers approach bulklike characteristics at a separation distance of ∼5 nm, the dynamics differ considerably. The complex association process is observed to be accompanied by several structural and dynamical changes of the system, such as large-scale correlated water density fluctuations, coupled conformational fluctuation of protein monomers, a dewettinglike transition with the change of intermonomeric distance RMM from ∼4 to ∼2 nm, orientation of monomers and hydrophobic hydration in the monomers. A quasistable, solvent-shared, protein monomer pair (SSPMP) forms at around 2 nm during association process which is a local free-energy minimum having ∼50–60% of native contacts. Simulations starting with arrangements sampled from the transition state (TS) of the dimer dissociation reveal that the final outcome depends on relative orientation of the backbone in the “hotspot” region.

Perfluorooctane Sulfonate (PFOS) Produces Dopaminergic Neuropathology in Caenorhabditis elegans

Perfluorooctane sulfonate (PFOS) has been widely utilized in numerous industries. Due to long environmental and biological half-lives, PFOS is a major public health concern. Although the literature suggests that PFOS may induce neurotoxicity, neurotoxic mechanisms, and neuropathology are poorly understood. Thus, the primary goal of this study was to determine if PFOS is selectively neurotoxic and potentially relevant to specific neurological diseases. Nematodes (Caenorhabditis elegans) were exposed to PFOS or related per- and polyfluoroalkyl substances (PFAS) for 72 h and tested for evidence of neuropathology through examination of cholinergic, dopaminergic, gamma-amino butyric acid (GABA)ergic, and serotoninergic neuronal morphologies. Dopaminergic and cholinergic functional analyses were assessed through 1-nonanol and Aldicarb assay. Mechanistic studies assessed total reactive oxygen species, superoxide ions, and mitochondrial content. Finally, therapeutic approaches were utilized to further examine pathogenic mechanisms. Dopaminergic neuropathology occurred at lower exposure levels (25 ppm, approximately 50 µM) than required to produce neuropathology in GABAergic, serotonergic, and cholinergic neurons (100 ppm, approximately 200 µM). Further, PFOS exposure led to dopamine-dependent functional deficits, without altering acetylcholine-dependent paralysis. Mitochondrial content was affected by PFOS at far lower exposure level than required to induce pathology (≥1 ppm, approximately 2 µM). Perfluorooctane sulfonate exposure also enhanced oxidative stress. Further, mutation in mitochondrial superoxide dismutase rendered animals more vulnerable. Neuroprotective approaches such as antioxidants, PFAS-protein dissociation, and targeted (mitochondrial) radical and electron scavenging were neuroprotective, suggesting specific mechanisms of action. In general, other tested PFAS were less neurotoxic. The primary impact is to prompt research into potential adverse outcomes related to PFAS-induced dopaminergic neurotoxicity in humans.

Force-dependent facilitated dissociation can generate protein-DNA catch bonds

Cellular structures are continually subjected to forces, which may serve as mechanical signals for cells through their effects on biomolecule interaction kinetics. Typically, molecular complexes interact via “slip bonds,” so applied forces accelerate off rates by reducing transition energy barriers. However, biomolecules with multiple dissociation pathways may have considerably more complicated force dependencies. This is the case for DNA-binding proteins that undergo “facilitated dissociation,” in which competitor biomolecules from solution enhance molecular dissociation in a concentration-dependent manner. Using simulations and theory, we develop a generic model that shows that proteins undergoing facilitated dissociation can form an alternative type of molecular bond, known as a “catch bond,” for which applied forces suppress protein dissociation. This occurs because the binding by protein competitors responsible for the facilitated dissociation pathway can be inhibited by applied forces. Within the model, we explore how the force dependence of dissociation is regulated by intrinsic factors, including molecular sensitivity to force and binding geometry, and the extrinsic factor of competitor protein concentration. We find that catch bonds generically emerge when the force dependence of the facilitated unbinding pathway is stronger than that of the spontaneous unbinding pathway. The sharpness of the transition between slip- and catch-bond kinetics depends on the degree to which the protein bends its DNA substrate. These force-dependent kinetics are broadly regulated by the concentration of competitor biomolecules in solution. Thus, the observed catch bond is mechanistically distinct from other known physiological catch bonds because it requires an extrinsic factor – competitor proteins – rather than a specific intrinsic molecular structure. We hypothesize that this mechanism for regulating force-dependent protein dissociation may be used by cells to modulate protein exchange, regulate transcription, and facilitate diffusive search processes.Statement of significanceMechanotransduction regulates chromatin structure and gene transcription. Forces may be transduced via biomolecular interaction kinetics, particularly, how molecular complexes dissociate under stress. Typically, molecules form “slip bonds” that dissociate more rapidly under tension, but some form “catch bonds” that dissociate more slowly under tension due to their internal structure. We develop a model for a distinct type of catch bond that emerges via an extrinsic factor: protein concentration in solution. Underlying this extrinsic catch bond is “facilitated dissociation,” whereby competing proteins from solution accelerate protein-DNA unbinding by invading the DNA binding site. Forces may suppress invasion, inhibiting dissociation, as for catch bonds. Force-dependent facilitated dissociation can thus govern the kinetics of proteins sensitive to local DNA conformation and mechanical state.