Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, δ-, and ϵ- subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type β-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

Co-Silencing of the Voltage-Gated Calcium Channel β Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor

The voltage-gated calcium channel (VGCC) β subunit (Cavβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavβ subunit gene of D. destructor, namely, DdCavβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCavβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCavβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavβ or HVA Cavα1 alone and co-silencing of the DdCavβ and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

A Suppressor Mutation in the β-Subunit Kis1 Restores Functionality of the SNF1 Complex in Candida albicans snf4 Δ Mutants

The highly conserved protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans , but it is not clear how it regulates its downstream targets in this fungus. We show that the repressor proteins Mig1 and Mig2 are phosphorylated also in cells lacking the catalytic α-subunit Snf1 of the SNF1 complex, but the amounts of both proteins were reduced in wild-type cells when glucose was replaced by alternative carbon sources, pointing to an indirect mechanism of regulation.

Involvement of Calcium-Dependent Pathway and β Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex

Introduction: Gain-of-function mutations in the L-type Ca2+ channel Cav1.2 cause Timothy syndrome (TS), a multisystem disorder associated with neurologic symptoms, including autism spectrum disorder (ASD), seizures, and intellectual disability. Cav1.2 plays key roles in neural development, and its mutation can affect brain development and connectivity through Ca2+-dependent and -independent mechanisms. Recently, a gain-of-function mutation, I1166T, in Cav1.2 was identified in patients with TS-like disorder. Its channel properties have been analyzed in vitro but in vivo effects of this mutation on brain development remain unexplored.Methods:In utero electroporation was performed on ICR mice at embryonic day 15 to express GFP, wild-type, and mutant Cav1.2 channels into cortical layer 2/3 excitatory neurons in the primary somatosensory area. The brain was fixed at postnatal days 14–16, sliced, and scanned using confocal microscopy. Neuronal migration of electroporated neurons was examined in the cortex of the electroporated hemisphere, and callosal projection was examined in the white matter and contralateral hemisphere.Results: Expression of the I1166T mutant in layer 2/3 neurons caused migration deficits in approximately 20% of electroporated neurons and almost completely diminished axonal arborization in the contralateral hemisphere. Axonal projection in the white matter was not affected. We introduced second mutations onto Cav1.2 I1166T; L745P mutation blocks Ca2+ influx through Cav1.2 channels and inhibits the Ca2+-dependent pathway, and the W440A mutation blocks the interaction of the Cav1.2 α1 subunit to the β subunit. Both second mutations recovered migration and projection.Conclusion: This study demonstrated that the Cav1.2 I1166T mutation could affect two critical steps during cerebrocortical development, migration and axonal projection, in the mouse brain. This is mediated through Ca2+-dependent pathway downstream of Cav1.2 and β subunit-interaction.

Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Characterisation of the redox centers of ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI–MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH. Graphical abstract

Modulatory Actions of the Glycine Receptor β Subunit on the Positive Allosteric Modulation of Ethanol in α2 Containing Receptors

Alpha1-containing glycine receptors (GlyRs) are major mediators of synaptic inhibition in the spinal cord and brain stem. Recent studies reported the presence of α2-containing GlyRs in other brain regions, such as nucleus accumbens and cerebral cortex. GlyR activation decreases neuronal excitability associated with sensorial information, motor control, and respiratory functions; all of which are significantly altered during ethanol intoxication. We evaluated the role of β GlyR subunits and of two basic amino acid residues, K389 and R390, located in the large intracellular loop (IL) of the α2 GlyR subunit, which are important for binding and functional modulation by Gβγ, the dimer of the trimeric G protein conformation, using HEK-293 transfected cells combined with patch clamp electrophysiology. We demonstrate a new modulatory role of the β subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2β heteromeric conformation. Indeed, while α2 was insensitive, α2β GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gβγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2β GlyRs. Mutations in an α2 GlyR co-expressed with the β subunit (α2AAβ) specifically blocked ethanol sensitivity, but not propofol potentiation. These results show a selective mechanism for low ethanol concentration effects on homomeric and heteromeric conformations of α2 GlyRs and provide a new mechanism for ethanol pharmacology, which is relevant to upper brain regions where α2 GlyRs are abundantly expressed.

Charcot–Marie–Tooth Disease With Episodic Rhabdomyolysis Due to Two Novel Mutations in the β Subunit of Mitochondrial Trifunctional Protein and Effective Response to Modified Diet Therapy

A 29-year-old female experienced chronic progressive peripheral neuropathy since childhood and was diagnosed with Charcot–Marie–Tooth disease (CMT) at age 15. She developed recurrent, fever-induced rhabdomyolysis (RM) at age 24. EMG studies showed decreased amplitude of compound muscle action potential, declined motor conductive velocity, and absence of sensor nerve action potential. Acylcarnitine analysis revealed elevated C16-OH, C18-OH, and C18:1-OH. Muscle biopsy showed scattered foci of necrotic myofibers invaded by macrophages, occasional regenerating fibers, and remarkable muscle fiber type grouping. Whole-exome sequencing identified two novel heterozygous mutations: c.490G>A (p.G164S) and c.686G>A (p.R229Q) in HADHB gene encoding the β-subunit of mitochondrial trifunctional protein (MTP). Reduction of long-chain fatty acid via dietary restrictions alleviated symptoms effectively. Our study indicates that the defect of the MTP β-subunit accounts for both CMT and RM in the same patient and expands the clinical spectrum of disorders caused by the HADHB mutations. Our systematic review of all MTPD patients with dietary treatment indicates that the effect of dietary treatment is related to the age of onset and the severity of symptoms.

Novel Binding Partners for CCT and PhLP1 Suggest a Common Folding Mechanism for WD40 Proteins with a 7-Bladed Beta-Propeller Structure

This study investigates whether selected WD40 proteins with a 7-bladed β-propeller structure, similar to that of the β subunit of the G protein heterotrimer, interact with the cytosolic chaperonin CCT and its known binding partner, PhLP1. Previous studies have shown that CCT is required for the folding of the Gβ subunit and other WD40 proteins. The role of PhLP1 in the folding of Gβ has also been established, but it is unknown if PhLP1 assists in the folding of other Gβ-like proteins. The binding of three Gβ-like proteins, TBL2, MLST8 and CDC20, to CCT and PhLP1, was demonstrated in this study. Co-immunoprecipitation assays identified one novel binding partner for CCT and three new interactors for PhLP1. All three of the studied proteins interact with CCT and PhLP1, suggesting that these proteins may have a folding machinery in common with that of Gβ and that the well-established Gβ folding mechanism may have significantly broader biological implications than previously thought. These findings contribute to continuous efforts to determine common traits and unique differences in the folding mechanism of the WD40 β-propeller protein family, and the role PhLP1 has in this process.