Binding Subunit Research Articles

Role of eukaryotic initiation factor 3 in the non‐canonical mechanism of Barley Yellow Dwarf Virus translation initiation

Plant viruses are a world‐wide threat both economically and to the food supply. Many of these are positive‐strand RNA viruses that have developed a myriad of non‐canonical translation initiation mechanisms. These unique mechanisms circumvent cap‐dependent translational controls and enable the viruses to outcompete host cell mRNA for the translation machinery. Many RNA viruses lack the canonical 5′ 7‐methylguanosine cap in their mRNAs, thereby allowing them to bypass canonical translation initiation. Instead, these mRNAs utilize structures in the 3′ untranslated region (UTR) of their mRNA known as a 3′ CITE (cap‐independent translation element) to drive non‐canonical translation initiation. Recently, we have shown that a 3′ CITE in Barley Yellow Dwarf Virus (BYDV), termed the 3′ BTE (BYDV 3′ CITE), can bind eukaryotic translation initiation factor (eIF) 4G, eIF4A, and ATP‐bound eIF4B in order to recruit the 40S ribosomal subunit and form a pre‐initiation complex (PIC) at the 3′ BTE. These eIFs cannot directly recruit the 40S subunit to the 5′ UTR of BYDV, but must instead transfer the 40S subunits to the 5′ end of the mRNA scanning, AUG detection, and translation elongation to proceed. This transfer process is aided by a long‐distance, ‘kissing‐loop’, base pairing interaction between stem‐loops in the 5′ and 3′ ends of BYDV. This kissing loop interaction is relatively weak, leading to the hypothesis that additional factors are required to create a stable 5′–3′ interaction capable of efficiently transferring the PIC. Data from pulldown experiments have shown that, in addition to eIF4G, almost every subunit of eIF3 is bound to the BTE. Single‐molecule fluorescence co‐localization experiments have shown that eIF3 specifically and significantly enhanced binding of 40S subunits to the BTE. Moreover, fluorescence anisotropy‐based binding studies have shown that eIF3 binds directly to both the BTE and the 5′ UTR of BYDV, that the BTE and 5′ UTR do not compete for binding to eIF3, and that the presence of the 40S subunit increases the affinity with which eIF3 binds to the BTE and 5′ UTR. Collectively, these data suggest that eIF3, the 40S subunit, the 3′ BTE, and the 5′ UTR are components of a single macromolecular complex. Based on this interpretation of our data, we propose that eIF3 bridges the 3′ and 5′ ends of BYDV and aids the kissing loop interaction in transferring the PIC from the 3′ to the 5′ end of BYDV.Support or Funding InformationGrant Support: NSF MCB‐1513737 (to DJG), NSF‐IGERT Fellowship 0965983 (to PP), and NIH GM 084288 (to RLG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Control of hyperglycemia in male mice by leflunomide: mechanisms of action.

p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKTS473/T308 and S6K1T389 phosphorylation but decreased S6S235/236 and IRS-1S1101 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on ob/ob and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in ob/ob and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKTS473/T308 phosphorylation in the fat and muscle of ob/ob mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity in vitro, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.

Increased Protein Cysteine Sulfonation with Heme Destruction of Mitochondrial Complex III Mediates Cardiac Reperfusion Injury

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage, which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide. The cascading reactive oxygen species can propagate and potentially induce protein cysteine sulfonation (PrCSO3H) of mitochondrial proteins. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of IR‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30 min of ischemia (coronary ligation) and 24 h of reperfusion in vivo. In the IR region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p<0.001). Visible spectroscopic analysis indicated that IR mediated destruction of heme b and hemes (c+c1) in the mitochondria, thus supporting IR‐induced impairment of the enzymatic activity of complex III. However, no significant impairment of complex III activity and heme destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min coronary ligation in vivo despite a decreased state 3 respiration detected in the ischemic mitochondria. LC‐MS/MS analysis showed formation of PrCSO3H, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondria from IR regions had intensely increased oxidative modification with S‐sulfonation level on the cysteine ligand (at C236) of 2Fe‐2S cluster of Rieske iron‐sulfur protein (uqcrfs1), thus impairing the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that IR‐mediated increased PrCSO3H of hinge protein at C65 (subunit 8, uqcrh) and cytochrome c1 subunit at C140 and C220, which weakened the interaction of hinge protein with cytochrome c1 and proper complex formation between cytochrome c1 and cytochrome c. LC‐MS/MS analysis further identified 4‐HNE (4‐hydroxylnonenal) adduct of cytochrome c1 at C140, suggesting involvement of IR‐induced lipid peroxidation in heme destruction and PrCSO3H of complex III. LC‐MS/MS analysis also showed that IR‐enhancing PrCSO3H of core 1 subunit (uqcrc1 at C69, C268, C380, C410, C445, C453) and core 2 subunit (uqcrc2 at C191), which may affect the binding of subunit 9 inhibitory peptide to core 1 and core 2 subunits and reactivate the MPP (matrix processing peptidase) activity of the core 1/core 2 subunits of mammalian complex III. LC‐MS/MS analysis of ischemic mitochondria has indicated a modest reduction from the basal level of complex III PrCSO3H detected in the mitochondria of sham control heart, suggesting that physiologic conditions of hypoxia during ischemia suppressed PrCSO3H, and physiologic conditions of hyperoxygenation during reperfusion mediated enhancement of complex III PrCSO3H. In conclusion, reperfusion‐mediated heme destruction with increased PrCSO3H controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction in the post‐ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may related to the preservation of functional integrity of heme groups and protein structure within mitochondrial complex III.Support or Funding InformationNHLBI/NIH, HL083237This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid Evolution of Bacterial Exotoxin B Subunits Independent of A subunits: Sialic Acid Binding Preferences Correlate with Host Range and Intrinsic Toxicity

Several well‐known AB bacterial toxins have cytotoxic A subunits that are delivered to the cytosol following B subunit binding to terminal sialic acid‐presenting glycans on host cell surfaces. Phylogenetic analysis of known and predicted B subunits in available genomes showed a poor correlation with that of associated A subunits, or even with that of the bacterial species phylogeny. In some cases, such as an exported protein of Yersinia pestis (YpB) there also appears to be no associated A subunit encoded in the genome. Y. pestis is the etiologic agent of Plague, responsible for major devastating epidemics in human history and is of global importance to public health and biodefense. Plague can never be eradicated because of the adaptability of the organism to infect more than 200 known host species. We noted that YpB shares 58% identity/79% similarity with the homopentameric B subunit of Subtilase cytotoxin, an AB5 toxin of E. coli, which also has 48% identity/68% similarity with that of the A2B5 S. Typhi typhoid toxin. We previously studied binding of such toxin B5 pentamers to a sialoglycan microarray and shown selective binding to different kinds of sialic acids that happen to be enriched in corresponding host cells, such as N‐acetylneuraminic acid (Neu5Ac; prominent in humans) or N‐glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Consistent with the much broader host range of Y. pestis, we have now found that YpB binds to essentially all types of sialic acids studied including Neu5Ac, Neu5Gc and their modified versions, and even to Kdn (2‐keto‐3‐deoxy‐D‐glycero‐D‐galacto‐nononic acid) a sialic acid prominent in reptiles and amphibians. Notably, YpB toxin alone can cause cytotoxicity that is abolished by mutations eliminating sialic acid recognition, suggesting that both uncontrolled cell proliferation and cell death might be mediated via lectin‐like cross‐linking of cell surface glycoproteins. These findings may help explain the wide host range of Y. pestis and could be important for understanding pathogenic mechanisms. Taken together, the data suggest Red Queen effects that are driving ongoing rapid evolution of both host sialic acids and pathogen toxin binding properties. Further studies of this clade of B5 toxins are revealing additional correlations of sialic acid binding preferences with host range and intrinsic toxicity, and molecular modeling studies are underway to define amino acid residues that determine binding differences.Support or Funding InformationNIH U01 CA199792This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence for direct interaction between RNA polymerase and the small ribosomal subunit.

In E. coli, RNA polymerase can directly bind ribosomes. This direct interaction may enable the coordination of the RNA polymerase and ribosome activities during transcription‐translation coupling. The coupling of both processes is exploited for gene regulation to enable immediate response to environmental changes.To establish transcription‐translation coupling, small ribosomal subunits have to be recruited to the nascent RNAs to initiate translation. Here, we report the recruitment of the small ribosomal subunit by RNA polymerase. Similar to the binding of ribosomes to RNA polymerase, the small ribosomal subunit binding to RNA polymerase is modulated by the conformation and functional state of both macromolecules. The RNA polymerase binding site on the small ribosomal subunit was identified by chemical crosslinking and is centered on the mRNA exit site of the small ribosomal subunit.Support or Funding InformationUniversity of California, Riverside (start‐up funding)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hybrids of carbon dots with subunit B of ricin toxin for enhanced immunomodulatory activity

Although Ricin toxin binding subunit B (RTB) can promote the activation of macrophages and modulate the cell-mediated immunity, its applications are severely limited due to the intrinsic properties of proteins, like poor stability and low efficacy of cellular uptake. In this work, the stable nanoparticles were prepared by supramolecular assembling of carbon dots (CDs) and RTB. The formed CDs-RTB possesses robust stability and can protect RTB against enzymatic hydrolysis. More importantly, CDs-RTB can promote macrophages proliferation, improve the generation of NO, IL-6 and TNF-α in RAW264.7 cells and increase the expression of mRNA, indicating the enhanced immunomodulatory activity of CDs-RTB. This work highlights the potential of using CDs as a simple and stable platform to assemble RTB and effectively promotes the application of RTB as the immunostimulant.

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATP-sensitive potassium channels (KATP) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic KATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic KATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

The Typhoid Toxin Produced by the Nontyphoidal Salmonella enterica Serotype Javiana Is Required for Induction of a DNA Damage Response In Vitro and Systemic Spread In Vivo.

ABSTRACTThe Salmonella cytolethal distending toxin (S-CDT), first described as the “typhoid toxin” in Salmonella enterica subsp. enterica serotype Typhi, induces DNA damage in eukaryotic cells. Recent studies have shown that more than 40 nontyphoidal Salmonella (NTS) serotypes carry genes that encode S-CDT, yet very little is known about the activity, function, and role of S-CDT in NTS. Here we show that deletion of genes encoding the binding subunit (pltB) and a bacteriophage muramidase predicted to play a role in toxin export (ttsA) does not abolish toxin activity in the S-CDT-positive NTS Salmonella enterica subsp. enterica serotype Javiana. However, S. Javiana strains harboring deletions of both pltB and its homolog artB, had a complete loss of S-CDT activity, suggesting that S. Javiana carries genes encoding two variants of the binding subunit. S-CDT-mediated DNA damage, as determined by phosphorylation of histone 2AX (H2AX), producing phosphorylated H2AX (γH2AX), was restricted to epithelial cells in S and G2/M phases of the cell cycle and did not result in apoptosis or cell death. Compared to mice infected with a ΔcdtB strain, mice infected with wild-type S. Javiana had significantly higher levels of S. Javiana in the liver, but not in the spleen, ileum, or cecum. Overall, we show that production of active S-CDT by NTS serotype S. Javiana requires different genes (cdtB, pltA, and either pltB or artB) for expression of biologically active toxin than those reported for S-CDT production by S. Typhi (cdtB, pltA, pltB, and ttsA). However, as in S. Typhi, NTS S-CDT influences the outcome of infection both in vitro and in vivo.

IRF9 and unphosphorylated STAT2 cooperate with NF-κB to drive IL6 expression

In response to IFNβ, the IL6 gene is activated, modestly at early times by ISGF3 (IRF9 plus tyrosine-phosphorylated STATs 1 and 2), and strongly at late times by U-ISGF3 (IRF9 plus U-STATs 1 and 2, lacking tyrosine phosphorylation). A classical IFN-stimulated response element (ISRE) at -1,513 to -1,526 in the human IL6 promoter is required. Pretreating cells with IFNβ or increasing the expression of U-STAT2 and IRF9 exogenously greatly enhances IL6 expression in response to the classical NF-κB activators IL1, TNF, and LPS. U-STAT2 binds tightly to IRF9, the DNA binding subunit of ISGF3, and also to the p65 subunit of NF-κB. Therefore, as shown by ChIP analyses, U-STAT2 can bridge the ISRE and κB elements in the IL6 promoter. In some cancer cells, the protumorigenic activation of STAT3 will be enhanced by the increased synthesis of IL6 that is facilitated by high expression of U-STAT2 and IRF9.

Cryo-electron microscopy structure of a human PRMT5:MEP50 complex.

Protein arginine methyl transferase 5 (PRMT5) is a signaling protein and histone modifying enzyme that is important in many cellular processes, including regulation of eukaryotic gene transcription. Reported here is a 3.7 Å structure of PRMT5, solved in complex with regulatory binding subunit MEP50 (methylosome associated protein 50, WDR77, p44), by single particle (SP) cryo-Electron Microscopy (cryo-EM) using micrographs of particles that are visibly crowded and aggregated. Despite suboptimal micrograph appearance, this cryo-EM structure is in good agreement with previously reported crystal structures of the complex, which revealed a 450 kDa hetero-octameric assembly having internal D2 symmetry. The catalytic PRMT5 subunits form a core tetramer and the MEP50 subunits are arranged peripherally in complex with the PRMT5 N-terminal domain. The cryo-EM reconstruction shows good side chain definition and shows a well-resolved peak for a bound dehydrosinefungin inhibitor molecule. These results demonstrate the applicability of cryo-EM in determining structures of human protein complexes of biomedical significance and suggests cryo-EM could be further utilized to understand PRMT5 interactions with other biologically important binding proteins and ligands.

The DNMT3A R882H mutant displays altered flanking sequence preferences.

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

Measuring the signs of the methyl 1H chemical shift differences between major and 'invisible' minor protein conformational states using methyl 1H multi-quantum spectroscopy.

Carr-Purcell-Meiboom-Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and 'invisible' minor states. The exchange rate(s) ([Formula: see text]), population(s) of the minor state(s) and the absolute value of the chemical shift difference [Formula: see text] (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of [Formula: see text] that is required to reconstruct the spectrum of the 'invisible' minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl [Formula: see text] CPMG experiment (Yuwen in Angew Chem 55:11490-11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of [Formula: see text] is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~18kDa cavity mutant of T4 Lysozyme ([Formula: see text]) and (2) the [Formula: see text]kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus ([Formula: see text]).

Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen.

ABSTRACTToxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA), the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast

Telomerase reverse transcriptase (TERT) and the non-coding telomerase RNA subunit (TR) constitute the core of telomerase. Here we now report that the putative F-box protein Pof8 is also a constitutive component of active telomerase in fission yeast. Pof8 functions in a hierarchical assembly pathway by promoting the binding of the Lsm2-8 complex to telomerase RNA, which in turn promotes binding of the catalytic subunit. Loss of Pof8 reduces TER1 stability, causes a severe assembly defect, and results in critically short telomeres. Structure profile searches identified similarities between Pof8 and telomerase subunits from ciliated protozoa, making Pof8 next to TERT the most widely conserved telomerase subunits identified to date.

Nonequilibrium Self-Assembly Dynamics of Icosahedral Viral Capsids Packaging Genome

The simplest icosahedral viruses can be viewed as nanometer-scaled protein shells called capsids encasing the genome. The packaging of genome is a crucial step for the survival of viruses, and it has to occur in a fast and accurate fashion. Surprisingly, little is known about the nonequilibrium dynamics that lead to infectious virions from their building blocks. Cooperative and nucleation-growth pathways have been identified in coarse-grained simulations, but even for the simplest viruses, experiments have failed so far to provide a clear-cut description of these phenomena. Here, we probed the self-assembly dynamics of icosahedral viral capsids packaging RNA genome or synthetic polyelectrolyte using time-resolved small-angle X-ray scattering with synchrotron source. We used the cowpea chlorotic mottle virus, a single-stranded RNA plant virus, whose capsid (28 nm in diameter) can self-assemble from and disassemble into dimeric subunits through long-lived intermediate species. By tuning the subunit-genome interactions through ionic strength, we found out that subunits and genome formed amorphous nucleoprotein complexes (NPCs) of ∼30 nm in size via a cooperative pathway. These NPCs in turn relaxed into virions via a nucleation-growth pathway by strengthening the subunit-subunit interactions through pH. The binding of subunits on genome could be as fast as 30 ms and the structural relaxation into virions could last several hours. Mean number of bound subunits as a function of time, binding and activation energies as well as apparent diffusion coefficient were estimated and the equilibrium species were imaged by cryo-transmission electron microscopy. Surprisingly, substituting synthetic polyelectrolyte to genome lowered the energy barrier to filled capsids. We propose a schematic free energy landscape that accounts for the observed pathways. Our results further point to the NPCs as checkpoints in the assembly pathway.

Clinical and Biophysical Characterization of a Mutation in the N-Helix Region of Cardiac Troponin C: Evidence for an Allosteric Mechanism of Contractile Dysfunction

Troponin is a heterotrimeric, Ca2+-binding protein responsible for regulating muscle contraction in the heart. Missense mutations in genes encoding the subunits of the cardiac troponin complex are associated with inherited dilated cardiomyopathy (DCM). However, the underlying molecular mechanisms of disease pathogenesis are not fully understood. Here, we report a clinical case of a 1-year-old female who presented with severe DCM and hypotonia. Whole exome sequencing revealed a previously unreported de novo heterozygous variant in exon 1 of the TNNC1 gene (c.12C>G), resulting in an Ile4Met mutation located in the N-Helix region of cardiac troponin C (cTnC). We utilized various biophysical and biochemical techniques to examine the molecular basis of this pathogenic variant and provide evidence for allosteric communication within the Ca2+-binding subunit of the troponin complex. In vitro extraction of native cTnC and reconstitution with either recombinant WT or I4M cTnC in cardiac muscle preparations revealed decreased Ca2+ sensitivity of isometric force development and slower kinetics of tension redevelopment (ktr) for I4M compared to WT cTnC. Steady-state fluorescence measurements on isolated cTnC using Bis-ANS indicated enhanced Ca2+ binding at the C-terminal domain of I4M compared to WT. Furthermore, cTnC-I4M displayed a smaller magnitude of Ca2+-induced hydrophobic exposure compared to WT. Finally, IANBD fluorescence (singly labeled at cysteine 84) titration studies revealed tighter binding of cardiac TnI to I4M compared to WT. Altogether, these results suggest that perturbed contractile kinetics and altered Ca2+-binding, perhaps by an allosteric mechanism, likely contribute to the contractile dysfunction observed in this proband. Experiments using solution NMR spectroscopy are currently underway to determine how a pathogenic mutation in the N-helix affects the overall structure and dynamics of cTnC. NIH-HL128683.

Tonically Active α2 Subunit-Containing Glycine Receptors Regulate the Excitability of Striatal Medium Spiny Neurons.

Medium spiny neurons (MSNs) of the dorsal striatum represent the first relay of cortico–striato–thalamic loop, responsible for the initiation of voluntary movements and motor learning. GABAergic transmission exerts the main inhibitory control of MSNs. However, MSNs also express chloride-permeable glycine receptors (GlyRs) although their subunit composition and functional significance in the striatum is unknown. Here, we studied the function of GlyRs in MSNs of young adult mice. We show that MSNs express functional GlyRs, with α2 being the main agonist binding subunit. These receptors are extrasynaptic and depolarizing at resting state. The pharmacological inhibition of GlyRs, as well as inactivation of the GlyR α2 subunit gene hyperpolarize the membrane potential of MSNs and increase their action potential firing offset. Mice lacking GlyR α2 showed impaired motor memory consolidation without any changes in the initial motor performance. Taken together, these results demonstrate that tonically active GlyRs regulate the firing properties of MSNs and may thus affect the function of basal ganglia.

Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments.

ABSTRACTLassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin.

We previously produced a heavy-chain-only antibody (Ab) VH domain (VHH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538-36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific VHHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the VHH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique VHHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 VHHs grouped into more than 20 different competition bins. The RTA-specific VHHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific VHHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.

A Supercluster of Neutralizing Epitopes at the Interface of Ricin's Enzymatic (RTA) and Binding (RTB) Subunits.

As part of an effort to engineer ricin antitoxins and immunotherapies, we previously produced and characterized a collection of phage-displayed, heavy chain-only antibodies (VHHs) from alpacas that had been immunized with ricin antigens. In our initial screens, we identified nine VHHs directed against ricin toxin’s binding subunit (RTB), but only one, JIZ-B7, had toxin-neutralizing activity. Linking JIZ-B7 to different VHHs against ricin’s enzymatic subunit (RTA) resulted in several bispecific antibodies with potent toxin-neutralizing activity in vitro and in vivo. JIZ-B7 may therefore be an integral component of a future VHH-based neutralizing agent (VNA) for ricin toxin. In this study, we now localize, using competitive ELISA, JIZ-B7’s epitope to a region of RTB’s domain 2 sandwiched between the high-affinity galactose/N-acetylgalactosamine (Gal/GalNAc)-binding site and the boundary of a neutralizing hotspot on RTA known as cluster II. Analysis of additional RTB (n = 8)- and holotoxin (n = 4)-specific VHHs from a recent series of screens identified a “supercluster” of neutralizing epitopes at the RTA-RTB interface. Among the VHHs tested, toxin-neutralizing activity was most closely associated with epitope proximity to RTA, and not interference with RTB’s ability to engage Gal/GalNAc receptors. We conclude that JIZ-B7 is representative of a larger group of potent toxin-neutralizing antibodies, possibly including many described in the literature dating back several decades, that recognize tertiary and possibly quaternary epitopes located at the RTA-RTB interface and that target a region of vulnerability on ricin toxin.