Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity

Supramodular nature of neuronal scaffolding proteins

Recent experimental evidence indicates that many scaffolding proteins localize at and make up synapses. These scaffolding proteins play important roles in both the maintenance of the architecture of synapses and the organization of neurotransmitters signaling complexes within synapses. Many scaffolding proteins are multidomain in nature, and these multimodular scaffold proteins often contain several repeat domains, which mainly mediate protein-protein interactions, arranged in tandem. The functional significances of such tandem arrangements of protein-protein interaction domains in scaffold proteins are largely unknown. In this thesis, I have presented the supramodular structure of the first two PDZ domains of PSD-95 (PDZ12). The structure of PDZ12 reveals that the two tandem PDZ domains are connected by a short and conserved inter-domain linker sequence ('KPPAE'). The PDZ 12 tandem structure further showes that the C-terminal peptide-binding groove on each of the two PDZ domains are aligned with a near parallel orientation. The supramodular arrangement of PSD-95 PDZ12 thereby affords the synergistic binding of the scaffold protein to multimerized carboxyl peptides. Therefore, my data provide a mechanistic explanation for PSD-95 mediated clustering of receptors and ion-channels. Indeed, abolition of the inter-domain orientation by insertion of nine amino acids ('GSSGSSGSS') into the linker region of PDZ1 and PDZ2 lowered the binding affinity of PDZ12 to its multimeric targets. I further went to demonstrate this insertion could decrease the clustering of potassium channel Kv4.1 in heterologous cells. The second part of this thesis addresses the supramodular nature of the tandem PDZ domains found in another neuronal scaffold protein, X11α. The three dimensional structures of the isolated PDZ domains as well as the tandem PDZ domains of X11α were determined. The structure shows that the first PDZ domain of X11α (PDZ1) binds to a carboxy1 peptide by recognizing only the last two amino acid residues. Such PDZ domain-mediated interaction with its target peptide is novel when compared to the canonical PDZ/target interaction. Most importantly, the structure of the tandem PDZ domain from X11α reveals that the C-terminus folds back and inserts into the target-recognition groove of PDZ1, thus keeping X11α in an auto-inhibited conformation. I further showed that the auto-inhibited conformation of the tandem PDZ domain from X11α was functionally important in synaptogenesis. The third part of this thesis describes the structural and biochemical basis of L27 domain-mediated supramodular complex organization. The three-dimensional structure of the tetrameric L27 domain composed of two L27S (L27 domain of SAP97) and two L27N (N-terminal L27 domain of mLin-2/CASK) were determined. The structure shows that each L27 domain contains three α-helices. The first two helices of each domain are packed against each other to form a four-helical bundle in the heterodimer. The third helix of each L27 domain forms another four-helical bundle that assembles the two heterodimers into a tetramer. The structure of the complex provides a mechanistic explanation for L27 domain-mediated polymerization of scaffold proteins. To systematically characterize L27 domain-mediated scaffold protein assembly, I have determined the three-dimensional structure of another L27-domain complex composed of L27M (L27 domain of Mals2/mLin-7) and L27C (the C-terminal L27 domain of mLin-2/CASK). Comparison of the two L27-domain complexes sheds light into the molecular specificity of L27 domain-mediated supramodular protein assembly. In summary, the work described in this thesis shows the supramolecular nature of neuronal scaffold proteins. This data provide many insights into the molecular mechanism of neuronal signal transduction

Sodium-glucose cotransporter 2 inhibitor may not prevent atrial fibrillation in patients with heart failure: a systematic review

Abstract Background Atrial fibrillation (AF) and heart failure (HF) frequently coexist because of their similar pathological basis. However, whether sodium-glucose cotransporter 2 inhibitor (SGLT2i), a novel class of anti-HF medication, decreases the risk of AF in HF patients remains unclear. Objectives The aim of this study was to assess the relationship between SGLT2i and AF in HF patients. Methods A meta-analysis of randomized controlled trails evaluating the effects of SGLT2i on AF in HF patients was performed. PubMed and ClinicalTrails.gov were searched for eligible studies until 27 November 2022. The risk of bias and quality of evidence were assessed through the Cochrane tool. Pooled risk ratio of AF for SGLT2i versus placebo in eligible studies was calculated. Results A total of 10 eligible RCTs examining 16,579 patients were included in the analysis. AF events occurred in 4.20% (348/8292) patients treated with SGLT2i, and in 4.57% (379/8287) patients treated with placebo. Meta-analysis showed that SGLT2i did not significantly reduce the risk of AF (RR 0.92; 95% CI 0.80–1.06; p = 0.23) in HF patients when compared to placebo. Similar results remained in the subgroup analyses, regardless of the type of SGLT2i, the type of HF, and the duration of follow-up. Conclusions Current evidences showed that SGLT2i may have no preventive effects on the risk of AF in patients with HF. Translational perspective Despite HF being one of the most common heart diseases and conferring increased risk for AF, affective prevention of AF in HF patients is still unresolved. The present meta-analysis demonstrated that SGLT2i may have no preventive effects on reducing AF in patients with HF. How to effectively prevent and early detect the occurrence of AF is worth discussing

Identification of genomic characteristics and selective signals in Guizhou black goat

Abstract Background Guizhou black goat is one of the indigenous black goat breeds in the southwest region of Guizhou, China, which is an ordinary goat for mutton production. They are characterized by moderate body size, black coat, favorite meat quality with tender meat and lower odor, and tolerance for cold and crude feed. However, little is known about the genetic characteristics or variations underlying their important economic traits. Results Here, we resequenced the whole genome of Guizhou black goat from 30 unrelated individuals breeding in the five core farms. A total of 9,835,610 SNPs were detected, and 2,178,818 SNPs were identified specifically in this breed. The population structure analysis revealed that Guizhou black goat shared a common ancestry with Shaanbei white cashmere goat (0.146), Yunshang black goat (0.103), Iran indigenous goat (0.054), and Moroccan goat (0.002). However, Guizhou black goat showed relatively higher genetic diversity and a lower level of linkage disequilibrium than the other seven goat breeds by the analysis of the nucleotide diversity, linkage disequilibrium decay, and runs of homozygosity. Based on F ST and θπ values, we identified 645, 813, and 804 selected regions between Guizhou black goat and Yunshang black goat, Iran indigenous goat, and cashmere goats. Combined with the results of XP-EHH, there were 286, 322, and 359 candidate genes, respectively. Functional annotation analysis revealed that these genes are potentially responsible for the immune response (e.g., CD28, CD274, IL1A, TLR2, and SLC25A31), humility-cold resistance (e.g., HBEGF, SOSTDC1, ARNT, COL4A1/2, and EP300), meat quality traits (e.g., CHUK, GAB2, PLAAT3, and EP300), growth (e.g., GAB2, DPYD, and CSF1), fertility (e.g., METTL15 and MEI1), and visual function (e.g., PANK2 and NMNAT2) in Guizhou black goat. Conclusion Our results indicated that Guizhou black goat had a high level of genomic diversity and a low level of linkage disequilibrium in the whole genome. Selection signatures were detected in the genomic regions that were mainly related to growth and development, meat quality, reproduction, disease resistance, and humidity-cold resistance in Guizhou black goat. These results would provide a basis for further resource protection and breeding improvement of this very local breed

A Genetically Modified Protein-Based Hydrogel for 3D Culture of AD293 Cells

<div><p>Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence ‘GRGDSP’ to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.</p></div

A schematic diagram of hydrogel formation.

<p>The Michael addition between maleimides of 4-armed-PEG-Mal and thiols in each mutant protein leads to the formation of 3D networks for hydrogelations. The blue balls represent TIP1 protein. The yellow balls represent amino acids that were replaced by cysteine in TIP1. The pink lines represent 4-armed-PEG-Mal.</p