Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Kinesin-1-dependent transport of the βPIX/GIT complex in neuronal cells

Proper targeting of the βPAK-interacting exchange factor (βPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the βPIX/GIT complex. Additionally, βPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of βPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited βPIX targeting the neurite tips in PC12 cells. Fur-thermore, truncated mutants of βPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of βPIX, and may provide new insights into βPIX/GIT complex-dependent neuronal pathophysiology.     

The roles of highly conserved,non‐catalytic residues in class A β‐lactamases

Evolution minimizes the number of highly conserved amino acid residues in proteins to ensure evolutionary robustness and adaptability. The roles of all highly conserved, non‐catalytic residues, 11% of all residues, in class A β‐lactamase were analyzed by studying the effect of 146 mutations on in cell and in vitro activity, folding, structure, and stability. Residues around the catalytic residues (second shell) contribute to fine‐tuning of the active site structure. Mutations affect the structure over the entire active site and can result in stable but inactive protein. Conserved residues farther away (third shell) ensure a favorable balance of folding versus aggregation or stabilize the folded form over the unfolded state. Once folded, the mutant enzymes are stable and active and show only localized structural effects. These residues are found in clusters, stapling secondary structure elements. The results give an integral picture of the different roles of essential residues in enzymes.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.     

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

Alzheimer''s disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

Deciphering the evolution of metallo-β-lactamases: A journey from the test tube to the bacterial periplasm

Understanding the evolution of metallo-β-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies.     

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

βII spectrin (SPTBN1): biological function and clinical potential in cancer and other diseases

βII spectrin, the most common isoform of non-erythrocyte spectrin, is a cytoskeleton protein present in all nucleated cells. Interestingly, βII spectrin is essential for the development of various organs such as nerve, epithelium, inner ear, liver and heart. The functions of βII spectrin include not only establishing and maintaining the cell structure but also regulating a variety of cellular functions, such as cell apoptosis, cell adhesion, cell spreading and cell cycle regulation. Notably, βII spectrin dysfunction is associated with embryonic lethality and the DNA damage response. More recently, the detection of altered βII spectrin expression in tumors indicated that βII spectrin might be involved in the development and progression of cancer. Its mutations and disorders could result in developmental disabilities and various diseases. The versatile roles of βII spectrin in disease have been examined in an increasing number of studies; nonetheless, the exact mechanisms of βII spectrin are still poorly understood. Thus, we summarize the structural features and biological roles of βII spectrin and discuss its molecular mechanisms and functions in development, homeostasis, regeneration and differentiation. This review highlight the potential effects of βII spectrin dysfunction in cancer and other diseases, outstanding questions for the future investigation of therapeutic targets. The investigation of the regulatory mechanism of βII spectrin signal inactivation and recovery may bring hope for future therapy of related diseases.     

Death-associated protein kinase 1 mediates Aβ42 aggregation-induced neuronal apoptosis and tau dysregulation in Alzheimer's disease

The aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils is a key pathological feature of Alzheimer''s disease (AD). An increasing amount of evidence suggests that oligomeric Aβ might be the major culprit responsible for various neuropathological changes in AD. Death-associated protein kinase 1 (DAPK1) is abnormally elevated in brains of AD patients and plays an important role in modulating tau homeostasis by regulating prolyl isomerase Pin1 phosphorylation. However, it remains elusive whether and how Aβ species influence the function of DAPK1, and whether this may further affect the function and phosphorylation of tau in neurons. Herein, we demonstrated that Aβ aggregates (both oligomers and fibrils) prepared from synthetic Aβ42 peptides were able to upregulate DAPK1 protein levels and thereby its function through heat shock protein 90 (HSP90)-mediated protein stabilization. DAPK1 activation not only caused neuronal apoptosis, but also phosphorylated Pin1 at the Ser71 residue, leading to tau accumulation and phosphorylation at multiple AD-related sites in primary neurons. Both DAPK1 knockout (KO) and the application of a specific DAPK1 inhibitor could effectively protect primary neurons against Aβ aggregate-induced cell death and tau dysregulation, corroborating the critical role of DAPK1 in mediating Aβ aggregation-induced neuronal damage. Our study suggests a mechanistic link between Aβ oligomerization and tau hyperphosphorylation mediated by DAPK1, and supports the role of DAPK1 as a promising target for early intervention in AD.     

Characterization and structural analyses of a novel glycosyltransferase acting on the β-1,2-glucosidic linkages

The IALB_1185 protein, which is encoded in the gene cluster for endo-β-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are β-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward β-1,2-glucooligosaccharides, and that the kinetic parameters for β-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the β-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites −1 and +1, whereas in the E343Q mutant–sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites −1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of β-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined β-1,2-glucooligosaccharide:d-glucoside β-d-glucosyltransferase as a systematic name and β-1,2-glucosyltransferase as an accepted name.     

3,4‐Dihydroxyphenylethanol and 3,4‐dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α‐synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect

Parkinson''s disease (PD) is a chronic multifactorial disease, whose etiology is not completely understood. The amyloid aggregation of α‐synuclein (Syn) is considered a major cause in the development of the disease. The presence of genetic mutations can boost the aggregation of the protein and the likelihood to develop PD. These mutations can lead to early onset (A30P, E46K, and A53T) or late‐onset (H50Q) forms of PD. The disease is also linked to an increase in oxidative stress and altered levels of dopamine metabolites. The molecular interaction of these molecules with Syn has been previously studied, while their effect on the pathological mutant structure and function is not completely clarified. By using biochemical and biophysical approaches, here we have studied the interaction of the familial variant E46K with two dopamine‐derived catechols, 3,4‐dihydroxyphenylacetic acid and 3,4‐dihydroxyphenylethanol. We show that the presence of these catechols causes a decrease in the formation of amyloid fibrils in a dose‐dependent manner. Native‐ and Hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) provide evidence that this effect is strongly conformation dependent. Indeed, these molecules interact differently with the interconverting conformers of Syn and its familial variant E46K in solution, selecting the most prone‐to‐aggregation one, confining it into an off‐pathway oligomer. These findings suggest that catechols could be a molecular scaffold for the design of compounds potentially useful in the treatment of Parkinson''s disease and related conditions.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

PI3KC2β inactivation stabilizes VE‐cadherin junctions and preserves vascular integrity

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3‐kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell–cell junctions by increasing Rab11‐dependent VE‐cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.