Search

Background: The novel coronavirus 2019 (COVID-19) infection has caused the global emergence of coronavirus in humans during the last 12 months. Till May 11, 2021, the confirmed global COVID-19 cases and deaths reached 158551526 and 3296855, respectively. Methods: Goblet cells and ciliated cells in the nose act as the initial infection site of SARS-CoV-2. Thus, mucus immunity is important to protect from infection. The outburst of SARS-CoV-2 infection can be halted only when an effective vaccine will be developed. Results: Globally, over 100 different vaccines are under investigation, including DNA vaccines, RNA vaccines, inactivated virus vaccines, adenovirus-based vaccines, recombinant/subunit protein vaccines, peptide vaccines, virus-like particles, etc. Inactivated virus vaccines and mRNA, and adenovirus-based vaccines have moved fast into clinical trials. Conclusion: Vaccines containing spike protein of SARS-CoV as subunit could effectively prevent binding of coronavirus to the host cell and membrane fusion. Thus, spike protein can be used as a major target for subunit vaccine preparation.

Voltage-gated Ca2+ channels are composed of a pore-forming α1 subunit and auxiliary β and α2δ subunits, which modulate Ca2+ current properties and channel trafficking. So far, the partial redundancy and specificity of α1 for α2δ subunits in the CNS have remained largely elusive. Mature spiral ganglion (SG) neurons express α2δ subunit isoforms 1, 2, and 3 and multiple Ca2+ channel subtypes. Differentiation and in vivo functions of their endbulb of Held synapses, which rely on presynaptic P/Q channels (Lin et al., 2011), require the α2δ3 subunit (Pirone et al., 2014). This led us to hypothesize that P/Q channels may preferentially co-assemble with α2δ3. Using a dissociated primary culture, we analyzed the effects of α2δ3 deletion on somatic Ca2+ currents (ICa ) of SG neurons isolated at postnatal day 20 (P20), when the cochlea is regarded to be mature. P/Q currents were the dominating steady-state Ca2+ currents (54% of total) followed by T-type, L-type, N-type, and R-type currents. Deletion of α2δ3 reduced P/Q- and R-type currents by 60 and 38%, respectively, whereas L-type, N-type, and T-type currents were not altered. A subset of ICa types was also analyzed in SG neurons isolated at P5, i.e., before the onset of hearing (P12). Both L-type and N-type current amplitudes of wildtype SG neurons were larger at P5 compared with P20. Deletion of α2δ3 reduced L-type and N-type currents by 23 and 44%, respectively. In contrast, small P/Q currents, which were just being up-regulated at P5, were unaffected by the lack of α2δ3. In summary, α2δ3 regulates amplitudes of L- and N-type currents of immature cultured SG neurons, whereas it regulates P/Q- and R-type currents at P20. Our data indicate a developmental switch from dominating somatic N- to P/Q-type currents in cultured SG neurons. A switch from N- to P/Q-type channels, which has been observed at several central synapses, may also occur at developing endbulbs of Held. In this case, reduction of both neonatal N- (P5) and more mature P/Q-type currents (around/after hearing onset) may contribute to the impaired morphology and function of endbulb synapses in α2δ3-deficient mice.

In the past studies, it is shown that cardiac troponin I (cTnI, encoded by TNNI3), as a cytoplasmic protein, is an inhibitory subunit in troponin complex, and involves in cardiomyocyte diastolic regulation. Here, we assessed a novel role of cTnI as a nucleoprotein. Firstly, the nuclear translocation of cTnI was found in mouse, human fetuses and rat heart tissues. In addition, there were differences in percentage of intranuclear cTnI in different conditions. Based on weighted gene co-expression network analyses (WGCNA) and verification in cell experiments, a strong expression correlation was found between TNNI3 and Atp2a2, which encodes sarco-endoplasmic reticulum Ca2+ ATPase isoform 2a (SERCA2a), and involves in ATP hydrolysis and Ca 2+ transient. TNNI3 gain and loss caused Atpa2a2 increase/decrease in a dose-dependent manner both in mRNA and protein levels, in vivo and in vitro. By using ChIP-sequence we demonstrated specific binding DNA sequences of cTnI were enriched in ATP2a2 promoter −239∼-889 region and the specific binding sequence motif of cTnI was analyzed by software as "CCAT", which has been reported to be required for YY1 binding to the promoter region of YY1-related genes. Moreover, it was further verified that pcDNA3.1 (−)-TNNI3 could express cTnI proteins and increase the promoter activity of Atp2a2 through luciferase report assay. In the end, we evaluated beat frequencies, total ATP contents, Ca 2+ transients in TNNI3-siRNA myocardial cells. These findings indicated, for the first time, cTnI may regulate Atp2a2 in cardiomyocytes as a co-regulatory factor and participate in the regulation of intracellular Ca ions.

The present study was focused to analyse and evaluate potential targets of NADPH Oxidase (NOX) at varying sub-units and co-relating their interactions with apocynin to identify appropreiate mechanism of inhibition for NOX by applying specific facets of computational biology . Active site molecular docking analysis was carried for apocynin against various subunits of NOX and comparatively analysise the binding affinity profiles which were obtained when apocynin was docked with p47phox complete subunit of NOX and all the other subunits.

In 2020 and 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, caused a global pandemic. Vaccines are expected to reduce the pressure of prevention and control, and have become the most effective strategy to solve the pandemic crisis. SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the surface spike (S) glycoprotein. In this study, a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells. The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody. Furthermore, the specific antibody titer and virus-neutralizing antibody (NAb) titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)3 and CpG. Our results showed that the virus-NAb lasted for more than 6 months in mice. To summarize, we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris, which stimulates neutralizing and protective antibody responses. A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

In most eukaryotes, the largest subunit of RNAPII, Rpb1, contains a conserved carboxyterminal domain (CTD) containing a canonical structure of heptapeptide repeats. Two protein complexes of interest, Mediator and Integrator, are known to interact with this CTD in all eukaryotic models they have been described in to date. Recently, orthologs of Mediator and Integrator subunits have been identified within the ciliated protozoan Tetrahymena thermophila; one of the few eukaryotic lineages to lack a canonically organized CTD. To begin to characterize putative Mediator and Integrator complexes within T. thermophila, I engineered appropriate macronuclear tagging and knockout cassettes. Although the Tetrahymena MED31 ortholog was unable to rescue the slow growth phenotype of a yeast MED31 knockout, or co-purify with yeast Med8-TAP, I identified subunit Med3 as a member of the Med31 interactome in T. thermophila through tandem affinity purification coupled with mass spectrometry. I also targeted the Tetrahymena INTS6 locus for knockout as determined by colony PCR. If Mediator and Integrator exist in Tetrahymena despite its divergent CTD of Rpb1, perhaps these complexes have CTD-independent functions beyond what can be effectively studied using conventional model systems.

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096 coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for alpha-galactoside-terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90 degree twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting alpha-galactosides. The alpha-Gal epitope is present on wild-type pig cells and the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllAs high specificity for alpha-galactoside-containing ligands, and we show that PllA can be used to visualize alpha-Gal epitope on porcine tissues.

In certain biologically relevant computing scenarios, a neuron "pools" the outputs of multiple independent functional subunits, firing if any one of them crosses threshold. Recent studies suggest that active dendrites could provide the thresholding mechanism, so that both the thresholding and pooling operations could take place within a single neuron. A pooling neuron faces a difficult task, however. Dendrites can produce highly variable responses depending on the density and spatial patterning of their synaptic inputs, and bona fide dendritic firing may be very rare, making it difficult for a neuron to reliably detect when one of its many dendrites has "gone suprathreshold". Our goal has been to identify biological adaptations that optimize a neuron's performance at the binary subunit pooling (BSP) task. Katz et al. (2009) pointed to the importance of spine density gradients in shaping dendritic responses. In a similar vein, we used a compartmental model to study how a neuron's performance at the BSP task is affected by different spine density layouts and other biological variables. We found BSP performance was optimized when dendrites have (1) a decreasing spine density gradient (true for many types of pyramidal neurons); (2) low-to-medium resistance spine necks; (3) strong NMDA currents; (4) fast spiking Na+ channels; and (5) powerful hyperpolarizing inhibition. Our findings provide a normative account that links several neuronal properties within the context of a behaviorally relevant task, and thus provide new insights into nature's subtle strategies for optimizing the computing capabilities of neural tissue.

Muscle phosphorylase b kinase (PHK) deficiency is a rare mild metabolic disorder caused by mutations of the PHKA1 gene encoding the αM subunit of PHK. A 16-year-old boy experienced myalgia during the maximal multistage 20-m shuttle run test targeting the maximal oxygen consumption. Although an ischemic forearm exercise test was normal, a muscle biopsy revealed subsarcolemmal glycogen accumulation. He harbored a novel insertion mutation in the PHKA1 gene that resulted in premature termination of the αM subunit close to the C-terminus. Compared with previously reported cases, his reduction in PHK activity was relatively mild.

Malaria is a human disease caused by eukaryotic protozoan parasites of the Plasmodium genus. Plasmodium falciparum (Pf) causes the most lethal form of human malaria and is responsible for widespread mortality worldwide. Prefoldin is a hetero-hexameric molecular complex that binds and delivers unfolded proteins to chaperonin for correct folding. The prefoldin PFD6 is predicted to interact with merozoite surface protein-1 (MSP-1), a protein well known to play a pivotal role in erythrocyte binding and invasion by Plasmodium merozoites. We previously found that the Plasmodium falciparum (Pf) genome contains six prefoldin genes and a prefoldin-like gene whose molecular functions are unidentified. Here, we analysed the expression of PfPFD-6 during the asexual blood stages of the parasite and investigated its interacting partners. PfPFD-6 was found to be significantly expressed at the trophozoite and schizont stages. Pull down assays suggest PfPFD-6 interacts with MSP-1. In silico analysis suggested critical residues involved in the PfPFD-6-MSP-1 interaction. Our data suggest PfPFD-6 may play a role in stabilizing or trafficking MSP-1.

Aims COVID-19 pandemic caused by SARS-CoV-2 has become a public health crisis worldwide. In this study, we aimed at demonstrating the neutralizing potential of the IgY produced after immunizing chicken with a recombinant SARS-CoV-2 spike protein S1 subunit. Methods and Results E. coli BL21 carrying plasmid pET28a-S1 was induced with IPTG for the expression of SARS-CoV-2 S1 protein. The recombinant His-tagged S1 was purified and verified by SDS-PAGE, Western blot and biolayer interferometry (BLI) assay. Then S1 protein emulsified with Freund's adjuvant was used to immunize layer chickens. Specific IgY against S1 (S1-IgY) produced from egg yolks of these chickens exhibited a high titer (1:25,600) and a strong binding affinity to S1 (KD = 318 nmol L−1). The neutralizing ability of S1-IgY was quantified by a SARS-CoV-2 pseudotyped virus-based neutralization assay with an IC50 value of 0.99 mg ml−1. In addition, S1-IgY exhibited a strong ability in blocking the binding of SARS-CoV-2 S1 to hACE2, and it could partially compete with hACE2 for the binding sites on S1 by BLI assays. Conclusions We demonstrated here that after immunization of chickens with our recombinant S1 protein, IgY neutralizing antibodies were generated against the SARS-CoV-2 spike protein S1 subunit; therefore, showing the potential use of IgY to block the entry of this virus. Significance and Impact of the Study IgY targeting S1 subunit of SARS-CoV-2 could be a promising candidate for pre- and post-exposure prophylaxis or treatment of COVID-19. Administration of IgY-based oral preparation, oral or nasal spray may have profound implications for blocking SARS-CoV-2.

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

The natural mutant mouse rolling Nagoya is severely ataxic and frequently shows body roll-overs. A missense mutation was identified in Cacna1a, the gene encoding the α1 pore subunit of CaV2.1 type voltage-gated Ca2+ channels. We here discuss the main phenotypic and neuronal characteristics of this mutant, with relevance to the human neurological disorders associated with CaV2.1 dysfunction (‘Ca2+-channelopathies’).

SummaryA biofilm inhibiting mechanism operates in the cyanobacterium Synechococcus elongatus. Here, we demonstrate that the glycosyltransferase homolog, Ogt, participates in the inhibitory process – inactivation of ogt results in robust biofilm formation. Furthermore, a mutational approach shows requirement of the glycosyltransferase activity for biofilm inhibition. This enzyme is necessary for glycosylation of the pilus subunit and for adequate pilus formation. In contrast to wild-type culture in which most cells exhibit several pili, only 25% of the mutant cells are piliated, half of which possess a single pilus. In spite of this poor piliation, natural DNA competence was similar to that of wild-type, therefore, we propose that the unglycosylated pili facilitate DNA transformation. Additionally, conditioned medium from wild-type culture, which contains a biofilm inhibiting substance(s), only partially blocks biofilm development by the ogt-mutant. Thus, we suggest that inactivation of ogt affects multiple processes including production or secretion of the inhibitor as well as the ability to sense or respond to it.Originality-Significance StatementThe molecular mechanisms that underlie biofilm development in cyanobacteria are just emerging. Using the cyanobacterium S. elongatus as a model, we demonstrate that glycosylation of the pilus subunit is crucial for the biofilm self-suppression mechanism, however, it is dispensable for DNA competence.

Numerous studies using a combination of confocal microscopic- and pharmacological-based approaches have demonstrated that the actin cytoskeleton dynamically responds to pathogen infection. Here, we observed that phalloidin treatment induced actin nucleation, resulting in enhanced resistance of wheat against the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). To define the mechanism underpinning this process, we characterized a family of conserved actin-binding proteins, the actin related protein (ARP) family, which controls actin polymerization. Specifically, we identified and characterized a wheat ARPC gene (TaARPC5), which encodes a 136-amino acid protein containing a P16-Arc domain, the smallest subunit of the ARP2/3 complex. TaARPC5 mRNA accumulation was induced following the infection of plants with the avirulent Pst strain, and following the elicitation with flagellin (e.g., flg22) as well. Subcellular localization analysis revealed that TaARPC5 is primarily localized to the cortical actin cytoskeleton, and its precise cellular localizations suggest the proximity to processes correlated with the actin-organelle interface. Upon treatment with virulent Pst, TaARPC5-knockdown plants exhibited a significant reduction in the expression of PTI-specific mRNAs. Conversely, we observed enhanced induction of reactive oxygen species (ROS) accumulation and a decrease in TaCAT1 expression following infection with an incompatible Pst isolate. Together with yeast complementation assays, the current study demonstrates a role for TaARPC5 in resistance signaling in wheat against Pst infection by regulating the host actin cytoskeleton.

3’-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3’-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3’-endonuclease activity of human CPSF with purified proteins. This requires the sevensubunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

Background Chaperonin containing TCP1 subunit 7 (CCT7), a member of the chaperonin containing TCP1 complex (CCT), has been reported regulating the expression of many tumor-related proteins. In this study, we investigated the diagnostic and prognostic value of CCT7 expression for hepatocellular carcinoma (HCC). Methods We investigated the CCT7 expression in HCC in The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and our cohort. The diagnostic and prognostic value were verified by receiver operating characteristic curve (ROC) analysis and Kaplan-Meier analysis, respectively. The association between CCT7 expression with DNA methylation status was investigated in the TCGA database. Gene ontology (GO), The Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) analysis were employed to identify the potential pathway in which CCT7 is involved in tumorigenesis and progression. Results CCT7 expression in HCC was significantly higher than adjacent normal tissues, and elevated CCT7 expression correlated with tumor stages and tumor grade. Furthermore, the ROC curve showed CCT mRNA expression has a better diagnostic value for HCC with early-stage and low alpha-fetoprotein expression. Positive predictive value (PPV) of CCT7 was higher than alpha-fetoprotein both in the GEO and TCGA database. The Multivariate Cox Regression analysis of clinicopathologic characteristics revealed that both high mRNA and protein expression of CCT7 were independent risk factors for overall survival (OS) and recurrence-free survival (RFS). High DNA methylation of CpG site(cg19515186) was associated with low CCT7 expression and better OS in HCC. The GO, KEGG and GSEA analysis demonstrated that CCT7 mRNA expression was associated with Spliceosome signaling pathway. Conclusions The findings of this study demonstrated that CCT7 has diagnostic and prognostic value for HCC.

Background: How signals from activated angiotensin type-2 receptors (AT 2 R) mediate inhibition of sodium ion (Na + ) reabsorption in renal proximal tubule cells is currently unknown. Protein phosphatases including PP2A (protein phosphatase 2A) have been implicated in AT 2 R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT 2 R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT 2 R activation in normal 4- and 10-week-old control Wistar-Kyoto rats and 4-week-old prehypertensive and 10-week-old hypertensive spontaneously hypertensive rats. Methods and Results: In Wistar-Kyoto rats, direct renal interstitial administration of selective AT 2 R nonpeptide agonist Compound-21 (C-21) increased renal interstitial cyclic GMP (cGMP) levels, urine Na + excretion, and simultaneously increased PP2A activity ≈2-fold in homogenates of renal cortical tubules. The cyclic GMP and natriuretic responses were abolished by concurrent renal interstitial administration of protein phosphatase inhibitor calyculin A. In renal proximal tubule cells in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT 2 Rs. Calyculin A treatment abolished C-21-induced translocation of both AT 2 R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT 2 R solubilized from renal cortical homogenates demonstrated physical association of AT 2 R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in spontaneously hypertensive rats, administration of C-21 did not alter urine Na + excretion or PP2A activity and failed to translocate AT 2 Rs and PP2A subunits to apical plasma membranes. Conclusions: In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT 2 Rs during induction of natriuresis. This response is defective in prehypertensive and hypertensive spontaneously hypertensive rats, presenting a potential novel therapeutic target for treating renal Na + retention and hypertension.

Avian pathogenic Escherichia coli (APEC) is the bacterial pathogen of poultry colibacillosis, which causes significant economic losses to the poultry industry. The lack of an effective vaccine against multiple serotypes and the emergence of multi-resistant isolates have made the control of avian colibacillosis troublesome. To identify conserved potential vaccine candidates, 58 genomes of APEC were obtained (54 sequenced by our laboratory and 4 downloaded from NCBI). A reverse vaccinology (RV) method based on the pangenome - called Pan-RV analysis - was performed in APEC-protective protein mining for the first time. Finally, four proteins were selected, and their immunoreactivity with anti-O1, O2, and O78 serum was verified by western blotting. Our in silico method of analysis will pave the way for rapid screening of vaccine candidates and will lay the foundation for the development of a highly effective subunit vaccine controlling APEC infection.

Mitochondrial homeostasis is crucial for the function of pancreatic β-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in β-cells. IF1 knockdown in cultured INS-1E β-cells enhances glucose-induced insulin release. However, the role of IF1 in islet β-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1-/-) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1-/- mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1-/-, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within β-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1-/- β-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 β-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 β-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in β-cells. Therefore, inhibiting IF1 to improve β-cell function in patients can be a novel therapeutic strategy to treat diabetes.

Aims: We aimed to explore the role of nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (Nox4) in the regulation of hypercalciuria-induced renal oxidative damage and crystal depositions...

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62–RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62–RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.

Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation., Discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to generate stable high molecular weight complexes. SpiD7 inhibits TNFα-induced nuclear translocation of RELA resulting in the blockade of NF-kB gene transcription, through a previously unexplored modality.

Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.

β cell number is maintained mainly by cell proliferation and cell apoptosis. Protein kinase A (PKA) pathway is an important intracellular signalling-mediating β cell proliferation. However, the precise roles of PKA isoforms are not well-defined. We found that the RIIB subunit of PKA is expressed specifically by β cells of mouse and human islets. Sixty percent pancreatectomy caused increased β cell proliferation. Deletion of type IIB PKA by disruption of RIIB expression further promoted β cell proliferation, leading to enhanced β cell mass expansion. RIIB KO mice also showed increased insulin levels and improved glucose tolerance. Mechanistically, activation of type IIB PKA decreased Cyclin D1 levels and inhibition of RIIB expression increased Cyclin D1 levels. Consistently, activation of type IIB PKA inhibited cell cycle entry. These results suggest that type IIB PKA plays a pivotal role in β cell proliferation via regulating Cyclin D1 expression.

The bacterial ribosomal protein S15 is located in the platform, a functional region of the 30S ribosomal subunit. While S15 is critical for in vitro formation of E. coli small subunits (SSUs), it is dispensable for in vivo biogenesis and growth. In this work, a novel synergistic interaction between rpsO, the gene that encodes S15, and rnc (the gene that encodes RNase III), was uncovered in E. coli. RNase III catalyzes processing of precursor ribosomal RNA (rRNA) transcripts and thus is involved in functional ribosome subunit maturation. Strains lacking S15 (ΔrpsO), RNase III (Δrnc) or both genes were examined to understand the relationship between these two factors and the impact of this double deletion on rRNA processing and SSU maturation. The double deletion of rpsO and rnc partially alleviates the observed cold sensitivity of ΔrpsO alone. A novel 16S rRNA precursor (17S∗ rRNA) that is detected in free 30S subunits of Δrnc is incorporated in 70S-like ribosomes in the double deletion. The stable accumulation of 17S∗ rRNA suggests that timing of processing events is closely coupled with SSU formation events in vivo. The double deletion has a suppressive effect on the cell elongation phenotype of ΔrpsO. The alteration of the phenotypes associated with S15 loss, due to the absence of RNase III, indicates that pre-rRNA processing and improvement of growth, relative to that observed for ΔrpsO, are connected. The characterization of the functional link between the two factors illustrates that there are redundancies and compensatory pathways for SSU maturation.