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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.

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.

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.

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.

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.

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.

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.

Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate σ70-dependent gene transcription through interacting with σ70 region 4 and the zinc binding domain (ZBD) of E. coli RNA polymerase (EcoRNAP) β′ subunit simultaneously. Despite extensive biochemical and structural analyses were reported recently, the interactions of SspA with RNAP are not comprehensively understood. Here, we reprocessed our previous cryo-EM dataset of EcoRNAP-promoter open complex with SspA (SspA-RPo) and obtained a significantly improved density map. Unexpectedly, the new map showed that SspA interacts with both N-terminal helix of β′ subunit (β′ΝΤΗ) and ω subunit, which contributes to stabilize the SspA-EcoRNAP σ70 holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of β′ subunit from different classes of bacteria revealed that β′ΝΤΗ is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of β′ΝΤΗ and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and β′ΝΤΗ facilitates the transcription regulation of SspA. Together, our results provide a more comprehensive insight into the interactions between SspA and RNAP and their roles in bacterial transcription regulation.

Acinetobacter baumannii is an opportunistic multidrug-resistant pathogen that causes a significant mortality rate. The proteins containing Tetratrico Peptide Repeats (TPRs) are involved in the pathogenicity and virulence of bacteria and have different roles such as transfer of bacterial virulence factors to host cells, binding to the host cells and inhibition of phagolysosomal maturation. So, in this study, physicochemical properties of a new protein containing TPRs in A. baumannii which was named PcTPRs1 by this study were characterized and its 3D structure was predicted by in-silico tools. The protein B and T cell epitopes were mapped and its vaccine potential was in-silico and in-vivo investigated. Domain analysis indicated that the protein contains the Flp pilus assembly protein TadD domain which has three TPRs. The helix is dominant in the protein structure, and this protein is an outer membrane antigen which, is extremely conserved among A. baumannii strains; thus, has good properties to be applied as a recombinant vaccine. The best-predicted and refined model was applied in ligand-binding sites and conformational epitopes prediction. Based on epitope mapping results, several epitopes were characterized which could stimulate both immune systems. BLAST results showed the introduced epitopes are completely conserved among A. baumannii strains. The in-vivo analysis indicates that a 101 amino acid fragment of the protein which contains the best selected epitope, can produce a good protectivity against A. baumannii as well as the whole TPR protein and thus could be investigated as an effective subunit and potential vaccines.

NMDAR (N-methyl-d-aspartate receptor) consisted of GluN1 and GluN2, and/or GluN3 subunits. As the obligatory subunit of NMDAR, GluN1 contains variant N-terminal domain (NTD) and C-terminal domain (CTD). The CTD contains allosteric signal and mediates the metabotropic function of NMDAR, which has been confirmed by previous studies. However, the allosteric signaling mechanism of GluN1 CTD has not been studied. In our study, we found that GluN1 CTD could bind to the lipid bilayers and affect the antigen epitope of GluN1 C-terminal antibody, suggesting that membrane binding may determine the allosteric signal of GluN1 CTD. In addition, we discovered that the membrane binding of GluN1 CTD could be regulated by the phosphorylation of GluN1 CTD C1 region.

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

During HIV-1 infection, the envelope glycoprotein subunit gp41 folds into a six-helix bundle structure (6HB) formed by the interaction between its N-terminal (NHR) and C-terminal (CHR) heptad-repeats, promoting viral and cell membranes fusion. A highly preserved, hydrophobic pocket (HP) on the NHR surface is crucial in 6HB formation and, therefore, HP-binding compounds constitute promising therapeutics against HIV-1. Here, we investigated the conformational and dynamic properties of the HP using a rationally designed single-chain protein (named covNHR) that mimics the gp41 NHR structure. We found that the fluorescent dye 8-anilino-naphtalene- 1-sulfonic acid (ANS) binds specifically to the HP, suggesting that ANS derivatives may constitute lead compounds to inhibit 6HB formation. ANS shows different binding modes to the HP, depending on the occupancy of other NHR pockets. Moreover, in presence of a CHR peptide bound to the N-terminal pockets in gp41, two ANS molecules can occupy the HP showing cooperative behavior. This binding mode was assessed using molecular docking and molecular dynamics simulations. The results show that the HP is conformationally flexible and connected allosterically to other NHR regions, which strongly influence the binding of potential ligands. These findings could guide the development of small-molecule HIV-1 inhibitors targeting the HP., Spanish State Research Agency, SRA/ 10.13039/501100011033 (grants BIO2016-76640-R and PID2019.107515RB.C21), ERDF/ESF

Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. While most of these disease-linked variants cause loss-of-function due to misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Co-expression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Co-expression with Cx46 also produced channels with altered voltage-gating properties, particularly at negative potentials. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal domain, while also neutralizing the voltage-sensing residue D3 as well as residue E42 which participates in loop-gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts.Statement of significanceWe investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system and showed that T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Consistent with our experimental findings, all-atom equilibrium state molecular dynamics (MD) simulations of T39R show that R39 stabilized the open-state configuration of the N-terminal (NT) domain from an adjacent subunit. These results suggest that T39R causes disease by preventing the hemichannels from closing when present in the plasma membrane in the undocked state and provide an atomistic rationalization for the Cx50 disease-linked phenotype. They also expand our understanding of how connexin hemichannel channel gating is controlled.

Energy metabolism is essential for almost all organisms. At the molecular level, adenosine monophosphate activated protein kinase (AMPK) plays a vital role in cellular energy homeostasis. Its molecular characterization in invertebrates, including Daphnia pulex, and the understanding of its role in response to environmental contaminants is limited. In this study, three subunits of AMPK (AMPKα, β, and γ) were cloned in D. pulex, and assigned the GenBank accession numbers MT536758, MT536759, and MT536760, respectively. Their full lengths were 2,000, 1,384, and 2594 bp, respectively, and contained open reading frames of 526, 274, and 580 amino acids, respectively. Bioinformatic analysis revealed that the three AMPK subunits all have features representative of the AMPK superfamily, and were correspondingly clustered with each orthologue branch. The three AMPK subunit genes, AMPKα, β, and γ, had the highest similarity to those of other organisms at 82%, 94%, and 71%, respectively. Nanoplastics significantly increased AMPKα expression, but decreased that of AMPKβ and γ. These results identified AMPKα, β, and γ in D. pulex, and showed that they all encode proteins with conserved functional domains. This study provides basic information on how three types of AMPK in aquatic organisms respond to environmental contaminants.

We have previously reported an important role of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, in the support of critical oncogenic pathways required for oncogenesis and the malignant phenotype of pancreatic cancer. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor inhibits the protein-stability of PR55α via FBXL20, a p53-target gene that serves as a substrate recognition component of the SCF (Skp1_Cullin1_F-box) E3 ubiquitin ligase complex that promotes proteasomal degradation of its targeted proteins. Our studies show that inactivation of p53 by siRNA-knockdown, gene-deletion, HPV-E6-mediated degradation, or expression of the loss-of-function mutant p53R175H results in increased PR55α protein stability, which is accompanied by reduced protein expression of FBXL20 and decreased ubiquitination of PR55α. Subsequent studies demonstrate that knockdown of FBXL20 by siRNA mimics p53 deficiency, reducing PR55α ubiquitination and increasing PR55α protein stability. Functional tests indicate that ectopic p53R175H or PR55α expression results in an increase of c-Myc protein stability with concomitant dephosphorylation of c-Myc-T58, which is a PR55α substrate, whose phosphorylation otherwise promotes c-Myc degradation. A significant increase in anchorage-independent proliferation is also observed in normal human pancreatic cells expressing p53R175H or, to a greater extent, overexpressing PR55α. Consistent with the common loss of p53 function in pancreatic cancer, FBXL20 mRNA expression is significantly lower in pancreatic cancer tissues compared to pancreatic normal tissues and low FBXL20 levels correlate with poor patient survival. Collectively, these studies delineate a novel mechanism by which the p53/FBXL20 axis negatively regulates PR55α protein stability.

Aberrant activity of the histone methyltransferase polycomb repressive complex 2 (PRC2) has been linked to several cancers, with small-molecule inhibitors of the catalytic subunit of the PRC2 enhancer of zeste homologue 2 (EZH2) being recently approved for the treatment of epithelioid sarcoma (ES) and follicular lymphoma (FL). Compounds binding to the EED subunit of PRC2 have recently emerged as allosteric inhibitors of PRC2 methyltransferase activity. In contrast to orthosteric inhibitors that target EZH2, small molecules that bind to EED retain their efficacy in EZH2 inhibitor-resistant cell lines. In this paper we disclose the discovery of potent and orally bioavailable EED ligands with good solubilities. The solubility of the EED ligands was optimized through a variety of design tactics, with the resulting compounds exhibiting in vivo efficacy in EZH2-driven tumors.

Neuronal exosomes play a crucial role in intercellular communication in the brain and represent a promising biomarker for neurological diseases, including stroke. However, limited techniques are available for isolating neuronal exosomes due to their small number in the serum exosomes. Thus, the development of efficient tools with brain-specific markers is needed. Here, we show the optimization of an immunoaffinity assay-based isolation protocol for specific exosomes or neuronally derived exosomes (NDE). Our results demonstrated that one-micron functionalized magnetic beads successfully separated CD63+ and L1CAM+ exosomes from serum. The size and shape of exosomes or exosomes pulled by beads were confirmed by Dynamic light scattering and Transmission electron microscopy; also, beads were well resolved in conventional flow cytometry analysis, which revealed that CD63-pulled serum exosomes had 5% expression of L1CAM. Furthermore, transmission electron microscopy showed that exosomes eluted from magnetic beads retained their original size, shape, and form without any damage. Furthermore, we showed isolation of NDE using GluR2/3-capturing antibody (α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor) using an optimized immunoaffinity bead assay utilizing 100 µl serum of stroke patients or age-matched healthy group. GluR2/3-captured exosomes were confirmed by western blot analysis. The western blot analysis showed a significant increase in the 35KDa subunit of GluR2/3 receptor protein in the exosomes of stroke patients compared to the healthy group. In addition, the multimeric GluR2/3 receptor protein in exosomes was further validated by the presence of the GluR2 subunit. Thus, our study shows GluR3/2 may be an effective candidate to isolate neuronal exosomes.

Large-pore channels, including those formed by connexin, pannexin, innexin proteins, are part of a broad family of plasma membrane channels found in vertebrates and invertebrates, which share topology features. Despite their relevance in parasitic diseases such as Chagas and malaria, it was unknown whether these large-pore channels are present in unicellular organisms. We identified 14 putative proteins in Trypanosomatidae parasites as presumptive homologs of innexin proteins. All proteins possess the canonical motif of the innexin family, a pentapeptide YYQWV, and 10 of them share a classical membrane topology of large-pore channels. A sequence similarity network analysis confirmed their closeness to innexin proteins. A bioinformatic model showed that a homolog of Trypanosoma cruzi (T. cruzi) could presumptively form a stable octamer channel with a highly positive electrostatic potential in the internal cavities and extracellular entrance due to the notable predominance of residues such as Arg or Lys. In vitro dye uptake assays showed that divalent cations-free solution increases YO-PRO-1 uptake and hyperosmotic stress increases DAPI uptake in epimastigotes of T. cruzi. Those effects were sensitive to probenecid. Furthermore, probenecid reduced the proliferation and transformation of T. cruzi. Moreover, probenecid or carbenoxolone increased the parasite sensitivity to antiparasitic drugs commonly used in therapy against Chagas. Our study suggests the existence of innexin homologs in unicellular organisms, which could be protein subunits of new large-pore channels in unicellular organisms.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

The terminal oxidases of the oxidative phosphorylation pathway play a significant role in the survival and growth of M. tuberculosis, targeting these components lead to inhibition of M. tuberculosis. Many drug candidates targeting various components of the electron transport chain in M. tuberculosis have recently been discovered. The cytochrome bc1-aa3 supercomplex is one of the most important components of the electron transport chain in M. tuberculosis, and it has emerged as the novel target for several promising candidates. There are two cryo-electron microscopy structures (PDB IDs: 6ADQ and 6HWH) of the cytochrome bc1-aa3 supercomplex that aid in the development of effective and potent inhibitors for M. tuberculosis. In recent years, a number of potential candidates targeting the QcrB subunit of the cytochrome bc1 complex have been developed. In this review, we describe the recently identified inhibitors that target the electron transport chain's terminal oxidase enzyme in M. tuberculosis, specifically the QcrB subunit of the cytochrome bc1 complex.

HIV-Associated Dementia (HAD) is a significant comorbidity that many HIV-patients face. Our study utilized QIAGEN Ingenuity Pathway Analysis (IPA) to identify and analyze molecular profiles and pathways underlying nicotine’s impact on HAD pathology. The Qiagen Knowledge Base (QKB) defines HAD as “Dementia associated with acquired immunodeficiency syndrome (disorder).” Although much remains unknown about HAD pathology, the curated research findings from the QKB shows 5 upregulated molecules that are associated with HAD + : CCL2 (Chemokine (C–C motif) ligand 2), L-glutamic acid, GLS (Glutaminase), POLG (DNA polymerase subunit gamma), and POLB (DNA polymerase subunit beta). The current study focused on these 5 HAD pathology molecules as the phenotype of interest. The Pathway Explorer tool of IPA was used to connect nicotine-associated molecules with the 5 HAD associated molecules (HAD pathology molecules) by connecting 29 overlapping molecules (including transcription regulators, cytokines, kinases, and other enzymes/proteins). The Molecule-Activity-Predictor (MAP) tool predicted nicotine-induced activation of the HAD pathology molecules indicating the exacerbation of HAD. However, alternative pathways with more holistic representations of molecular relationships revealed the potential of nicotine as a neuroprotective treatment. It was found that concurrent with nicotine treatment the individual inactivation of several of the intermediary molecules in the holistic pathways caused the downregulation of the HAD pathology molecules. These findings reveal that nicotine may have therapeutic properties for HAD when given alongside specific inhibitory drugs for one or more of the identified intermediary molecules.

We have previously shown that the αvβ6 integrin plays a key role in promoting prostate cancer (PrCa) and it can be transferred to recipient cells via small extracellular vesicles (sEVs). Furthermore, we have reported in a proteomic analysis that αvβ6 integrin down-regulation increases the expression of IFIT3 (interferon induced protein with tetratricopeptide repeats 3) in PrCa cells and their derived sEVs. IFIT3 is a protein well known for being an antiviral effector, but recently its role in cancer has also been elucidated. To study the relationship between IFIT3 and STAT1 (signal transducer and activator of transcription 1), an upstream regulator of IFIT3, in PrCa cells and their released sEVs, we used CRISPR/Cas9 techniques to down-regulate the expression of the β6 integrin subunit, IFIT3 or STAT1. Our results show that IFIT3 and STAT1 are highly expressed in PrCa cells devoid of the β6 integrin subunit. However, IFIT3 but not STAT1, is present in sEVs derived from PrCa cells lacking the β6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1 but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. Overall, these results provide new insights into the intrinsic role of IFIT3 as a regulator of STAT1 expression in sEVs and in intercellular communication in PrCa.

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein–DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs. ispartof: AMERICAN JOURNAL OF HUMAN GENETICS vol:108 issue:11 pages:2130-2144 ispartof: location:United States status: published

Cereal grain-derived protein hydrolysates exert beneficial effects for human health, thus utilization of by-products with high protein content has drawn attention. In this study, hot water extraction of rice husk has been optimized to obtain protein hydrolysate with the highest anticancer activity. The optimum protein value was obtained as 2.40 g/L corresponding to 43.2 g protein/kg dried rice husk under the conditions of 60 degrees C, 2.0 mL/min flow rate and pH 10.0. The protein hydrolysate was encapsulated with chitosan, where the mean particle size of protein hydrolysate (0.3 %) loaded chitosan nanoparticles was 256.4 +/- 33. 4 nm with 89 % encapsulation efficiency and a 65% release at the end of 6 days. Cytotoxicity assays showed that the lowest cell viabilities have been achieved with A549 and MCF7 cells with IC50 values of 1.98 and 3.58 mu g/mL, respectively, whereas nuclear fragmentation and apoptotic bodies were observed through Hoechst 33342 staining. The cytotoxic effect might be associated with the wide variety of peptide/protein subunits ranging from 10 kDa to more than 180 kDa in the protein hydrolysate of rice husk. (C) 2021 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved., Kocaeli University [KOU-BAP-2016-081, KOU-BAP-2018-149], The research funds provided by Kocaeli University (KOU-BAP-2016-081 and KOU-BAP-2018-149) are highly appreciated. Special thanks to Mr. Ahmet Yilmaz who provided the rice husk from Ozserhat Gida Sanayi Tic. Ltd. Sti, Edirne, Turkey. We are grateful to Dilan Yerli for helping with SDS-PAGE, Prof. Dr. Kemal Sami Korkmaz for providing access to his lab at Ege University and Pelin Saglam-Metiner for her contributions to fluorescent staining assays.

Cancer cells release a variety of factors that contribute to the alteration of proximal and distal tissues to promote metastasis. Recent studies have demonstrated that aggressive cancer cells release extracellular vesicles with higher protein content and in excess than extracellular vesicles isolated from patients with less aggressive disease or healthy individuals. We found that melanoma tumor-derived extracellular vesicles (TEV) downregulate type I interferon receptor subunit 1 (IFNAR1), suppress expression of the interferon stimulated gene cholesterol 25-hydroxylase (CH25H). Loss of CH25H is observed in the leukocytes from melanoma patients, which correlated with metastasis and poor survival. Similarly, mice also exhibit loss of IFNAR1 following TEV administration. Moreover, loss of CH25H increased TEV uptake and TEV-induced pre metastatic niche and lung metastasis. Use of the anti-hypertensive drug, reserpine, mimicked the effects of the CH25H product 25-hydroxycholesterol to suppress TEV uptake and TEV-mediated tumor growth, pre-metastatic niche formation, and lung metastasis. These results suggest the importance of CH25H in suppressing TEV mediate cancer progression and importance of developing strategies to suppress TEV uptake and TEV-mediated disease progression.

In SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19.

Hyperpolarization-gated, cyclic nucleotide-activated (HCN1-4) channels are inwardly rectifying cation channels that display voltage dependent activation and de-activation. Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies including the de novo HCN1 M305L variant. M305 is located in the S5 domain that is implicated in coupling voltage sensor domain movement to pore opening. This variant lacks voltage-dependent activation and de-activation and displays normal cation selectivity. To elucidate the impact of the mutation on the channel structure-function relations, molecular dynamics simulations of the wild type and mutant homotetramers were compared and identified a sulphur-aromatic interaction between M305 and F389 that contributes to the coupling of the voltage-sensing domain to the pore domain. To mimic the heterozygous condition as a heterotetrameric channel assembly, Xenopus oocytes were co-injected with various ratios of wild-type and mutant subunit cRNAs and the biophysical properties of channels with different subunit stoichiometries were determined. The results showed that a single mutated subunit was sufficient to significantly disrupt the voltage dependence of activation. The functional data were qualitatively consistent with predictions of a model that assumes independent activation of the voltage sensing domains allosterically controlling the closed to open transition of the pore. Overall, the M305L mutation results in an HCN1 channel that lacks voltage dependence and facilitates excitatory cation flow at membrane potentials that would normally close the channel. Our findings provide molecular insights into HCN1 channels and reveal the structural and biophysical basis of the severe epilepsy phenotype associated with the M305L mutation.

Non-SMC condensin I complex subunit D2 (NCAPD2) is one of the three non-SMC subunits in condensin I. Previous studies have shown that NCAPD2 plays an important role in the chromosome condensation and segregation. However, its role in the development of colorectal cancer (CRC) and specific molecular mechanisms still need to be further studied. Here we show that NCAPD2 inhibits autophagy and blocks autophagic flux via Ca2+/CAMKK/AMPK/mTORC1 pathway and PARP-1/SIRT1 axis. NCAPD2 acts as a tumor promoter both in vitro and in vivo. NCAPD2 knockout suppresses colorectal cancer development in AOM/DSS induced mice model. Therefore, our findings support a role for NCAPD2 in autophagy to promote CRC development and highlight NCAPD2 as a potential target for CRC therapy.

Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.

The funny current, and the HCN channels that form it, are affected by the direct binding of cyclic nucleotides. Binding of these second messengers causes a depolarizing shift of the activation curve, which leads to greater availability of current at physiological membrane voltages. This review outlines a brief history on this regulation and provides some evidence that other cyclic nucleotides, especially cGMP, may be important for the regulation of the funny channel in the heart. Current understanding of the molecular mechanism of cyclic nucleotide regulation is also presented, which includes the notions that full and partial agonism occur as a consequence of negatively cooperative binding. Knowledge gaps, including a potential role of cyclic nucleotide-regulation of the funny current under pathophysiological conditions, are included. The work highlighted here is in dedication to Dario DiFrancesco on his retirement.

Telomeres protect chromosome ends from degradation. Telomere length (TL) can be altered by aging and environmental stress. Shortened TL has been observed in peripheral blood leukocytes of alcohol dependent subjects and ethanol-exposed somatic cells. To understand the impact of ethanol on telomeres in pluripotent stem cells, we investigated the influence of ethanol on TL and the expression of six Shelterin complex subunit or telomere-regulating genes (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) in human embryonic stem cells (hESCs), which were exposed to 0, 25, 50, or 100 mM of ethanol for 3, 7, or 14 days. Ethanol-induced TL and Shelterin complex subunit gene expression changes were examined by quantitative polymerase chain reactions. Two-way ANOVA tests indicated that TL variation and expression changes of four associated Shelterin complex subunit genes (POT1, TPP1, TIN2, and TRF2) were mainly dependent on the length of ethanol exposure, while TRF1 and RAP1expression was influenced by ethanol concentration, exposure time, and the interaction of ethanol concentration and exposure time. Tukey's multiple comparison tests showed that TL and the expression of POT1, RAP1, TIN2, TPP1, and TRF1 were decreased after a 7-day (versus a 3-day) ethanol exposure. However, the decreased expression of all six Shelterin complex subunit genes was recovered and TL was not further shortened after a 14-day (versus a 7-day) ethanol exposure, likely due to the adaptation of hESCs to ethanol-induced stress. Our study provided further evidence that TL is regulated and maintained by telomere-regulating genes in stem cells under ethanol stress.

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as “methylation”). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

Thymic stromal lymphopoietin (TSLP) is a pro-inflammatory cytokine with important pathological roles in Asthma bronchiale, malignant tumours and other diseases. The heterodimeric human TSLP receptor (hTSLPR) consists of the TSLP-binding subunit (TSLPRα) and the IL-7Rα-subunit. We studied the properties of hTSLP variants with mutations in their bipartite interaction interface towards IL-7Rα. One mutant (T46D/K101D) showed only mild impairment in receptor affinity but a massive reduction in biological activity. To facilitate the future development of hTSLP mutants with drug properties, we have devised a eukaryontic cytokine display assay with activity read-out and intrinsic genotype-phenotype coupling.

The small subunit, ssPOXA3a/b, and the large subunit, POXA3, are indispensable components of typical heterodimeric laccase (Lacc2) in white rot fungi. However, the enzymatic and biological functions of ssPOXA3a/b remain unclear. The present study revealed that neither ssPOXA3a nor ssPOXA3b per se has a catalytic ability, whereas their combination with POXA3 (and especially ssPOXA3b) enhances the activity, thermostability, and pH stability of POXA3. In Pleurotus eryngii var. ferulae, there was no regulatory relationship between ssPOXA3a/b and POXA3 at the transcriptional level. However, sspoxa3a/b overexpression had a negative feedback effect on lacc6 transcription. By contrast, poxa3 transcripts had no effect on any other laccase isoenzyme. Overexpression of sspoxa3a/b resulted in small fungal pellets, thin mycelial walls, and facilitated laccase secretion. However, poxa3 overexpression had no influence on pellet morphology. Collectively, this work elucidated the functions of ssPOXA3a/b and laid an empirical foundation for the development of high-yield laccase.

Post-transcriptional modifications affect tRNA biology and are closely associated with human diseases. However, progress on the functional analysis of tRNA modifications in metazoans has been slow because of the difficulty in identifying modifying enzymes. For example, the biogenesis and function of the prevalent N2-methylguanosine (m2G) at the sixth position of tRNAs in eukaryotes has long remained enigmatic. Herein, using a reverse genetics approach coupled with RNA-mass spectrometry, we identified that THUMP domain-containing protein 3 (THUMPD3) is responsible for tRNA: m2G6 formation in human cells. However, THUMPD3 alone could not modify tRNAs. Instead, multifunctional methyltransferase subunit TRM112-like protein (TRMT112) interacts with THUMPD3 to activate its methyltransferase activity. In the in vitro enzymatic assay system, THUMPD3–TRMT112 could methylate all the 26 tested G6-containing human cytoplasmic tRNAs by recognizing the characteristic 3′-CCA of mature tRNAs. We also showed that m2G7 of tRNATrp was introduced by THUMPD3–TRMT112. Furthermore, THUMPD3 is widely expressed in mouse tissues, with an extremely high level in the testis. THUMPD3-knockout cells exhibited impaired global protein synthesis and reduced growth. Our data highlight the significance of the tRNA: m2G6/7 modification and pave a way for further studies of the role of m2G in sperm tRNA derived fragments.

Complexes of p24 proteins act as cargo receptors for the transport of COPII vesicles from the endoplasmic reticulum (ER). The major cargos of p24 complexes are hydrophilic proteins tethered to the ER membrane via a covalently attached glycosylphosphatidylinositol (GPI) or fatty acid. Each p24 complex is known to contain members from all four p24 subfamilies (p24α, p24β, p24γ and p24δ). However, it remains unclear how the cargo specificities of p24 complexes are influenced by member stoichiometry. Here, we report the subunit compositions of mammalian p24 complexes involved in the transport of GPI-anchored proteins and Wnt1. We show that at least one p24α is required for the formation of p24 complexes and that a p24 complex consisting of p24α2, p24β1, p24γ2 and p24δ1 is required for the efficient transport of GPI-anchored proteins. On the other hand, a p24 complex containing p24α2, p24α3, p24β1, p24γ and p24δ1 is involved in the transport of Wnt1. Further, interactions between p24α2 and p24α3 are critical for Wnt1 transport. Thus, p24α and p24γ subfamily members are important for cargo selectivity. Lastly, our data fit with an octamer, rather than a tetramer, model of p24 complexes, where each complex consists of two proteins from each p24 subfamily.