Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter’s activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

A mutation associated with Charcot-Marie-Tooth disease enhances the formation of stable dynamin 2 complexes in cells

Mutations in dynamin 2 (DNM2) have been associated with two distinct motor disorders, Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). The majority of these mutations are clustered in the pleckstrin homology domain (PHD) which engage in intramolecular interactions that suppress dynamin self-assembly and GTPase activation. CNM mutations in the PHD interferes with these intramolecular interactions, thereby blocking the formation of the auto-inhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for lipid PIP2 binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations cause either one disease or the other, despite their close proximity within the PHD sequence. We show that at least one CMT-causing mutant, lacking residues 555DEE557 (∆DEE), displays this inability to undergo auto-inhibition as observed in CNM-linked mutants. This ∆DEE deletion mutant induces the formation of abnormally large cytoplasmic inclusions similar to those observed for CNM-linked mutant R369W. We also found substantially reduced migration from the membrane of the ∆DEE deletion mutant. These findings call into question the molecular mechanism currently believed to underlie the absence of pathogenic overlap between DNM2-dependent CMT and CNM.

The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo

Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. In this study we found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. We demonstrate that the schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, we show that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, we employed epistasis experiments in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. We propose that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.

[CASE REPORT] Homozygous N-terminal missense variant in PLEKHG5 associated with intermediate CMT: a case report

Mutations in PLEKHG5, a pleckstrin homology domain containing member of the GEF family, are associated with distal spinal muscular atrophy and intermediate Charcot-Marie-Tooth disease. Here, we describe an isolated case with distal intermediate neuropathy with scapular winging. By whole exome sequencing, we identified the homozygous PLEKHG5 Arg97Gln missense mutation, located in the N-terminal region of the protein. This mutation resides between a zinc-finger motif and a RBD domain, involved in binding rnd3, a RhoA effector protein. We conclude that based on the characteristic phenotype presented by the patient and the supportive genetic findings, the PLEKHG5 mutation is the causative variant.

Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

The Sirenic Links between Diabetes, Obesity, and Bladder Cancer

There is considerable evidence of a positive association between the incidence of type 2 diabetes mellitus (T2DM) and obesity with bladder cancer (BCa), with the link between T2DM and obesity having already been established. There also appear to be potential associations between Pleckstrin homology domain containing S1 (PLEKHS1) and the Insulin-like Growth Factor (IGF) axis. Seven literature searches were carried out to investigate the backgrounds of these potential links. PLEKHS1 is a candidate biomarker in BCa, with mutations that are easily detectable in urine and increased expression seemingly associated with worse disease states. PLEKHS1 has also been implicated as a potential mediator for the onset of T2DM in people with obesity. The substantial evidence of the involvement of IGF in BCa, the role of the IGF axis in obesity and T2DM, and the global prevalence of T2DM and obesity suggest there is scope for investigating the links between these components. Preliminary findings on the relationship between PLEKHS1 and the IGF axis signal possible associations with BCa progression. This indicates that PLEKHS1 plays a role in the pathogenesis of BCa that may be mediated by members of the IGF axis. Further detailed research is needed to establish the relationship between PLEKHS1 and the IGF axis in BCa and determine how these phenomena overlap with T2DM and obesity.

Disruption of the Serine/Threonine Kinase Akt Gene Affects Ovarian Development and Fecundity in the Cigarette Beetle, Lasioderma serricorne

Serine/threonine kinase Akt, an important component of the insulin signaling pathway, plays an essential role in many physiological processes. In this study, we identified and characterized an Akt gene (designated LsAkt) from the cigarette beetle, Lasioderma serricorne. LsAkt contains a 1614 bp open reading frame encoding a 537 amino acid protein that possesses a conserved pleckstrin homology domain and a serine/threonine kinase domain. The expression of LsAkt was high in pupal stages and peaked in day-4 female pupae. In adult tissues, LsAkt was highly expressed in the thorax, ovary, and midgut. The expression of LsAkt was induced by methoprene or bovine insulin in vivo, but significantly decreased by 20-hydroxyecdysone. RNA interference-mediated knockdown of LsAkt resulted in severely blocked ovarian development and reduced fecundity and hatchability. The vitellogenin (Vg) content and juvenile hormone (JH) titers of LsAkt-depletion beetles were decreased, and expressions of Vg and four JH signaling and biosynthetic genes were significantly decreased. Silencing of LsAkt reduced the amounts of glucose, glycogen, and trehalose in female adults and affected the expressions of seven key carbohydrate metabolic genes. Taken together, it is inferred that Akt implicates in L. serricorne reproduction by modification of Vg synthesis, juvenile hormone production and carbohydrate metabolism.

Trypanosome morphogenesis involves recruitment of a pleckstrin homology domain protein by an orphan kinesin to the microtubule quartet

ABSTRACTKinesins are motor proteins found in all eukaryotic lineages that move along microtubule tracks to mediate numerous cellular processes such as mitosis and intracellular transport of cargo. In trypanosomatids, the kinesin protein superfamily has undergone a prominent expansion, giving these protists one of the most diverse kinesin repertoires. This has led to the emergence of two trypanosomatid-restricted groups of kinesins. Here, we characterize in Trypanosoma brucei TbKifX2, a hitherto orphaned kinesin that belongs to one of these groups. Representing a rare instance, TbKifX2 tightly interacts with TbPH1, a kinesin-like protein with an inactive motor domain. TbPH1 is named after a pleckstrin homology (PH) domain present within its carboxy-terminal tail. TbKifX2 recruits TbPH1 to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that is part of the multipartite flagellum attachment zone (FAZ) and extends from the basal body to the anterior of the cell body. The proximal proteome of TbPH1 is comprised of four proteins that localize to the FAZ, consistent with the notion that the TbKifX2/TbPH1 complex are the first identified proteins to bind the MtQ along its whole length. Simultaneous ablation of both TbKifX2 and TbPH1 leads to the formation of prominent protrusions from the cell posterior. Thus, these two trypanosomatid-restricted proteins, which specifically localize to the MtQ in a microtubule-rich cell, appear to be contributors to morphogenesis in T. brucei.IMPORTANCETrypanosomatids are a group of unicellular parasites that infect a wide range of hosts from land plants to animals. They are also eukaryotes that have been shaped by prolonged independent evolution since this domain of life has radiated from a common ancestor almost 2 billion years ago. Thus, any resulting unique biological properties can be potentially exploited for treatment of infectious diseases caused by trypanosomatids. The cytoskeleton of trypanosomatids represents an ancient organelle that has undergone such modification. Here, we show that two trypanosomatid-specific proteins named TbPH1 and TbKifX2 form a complex that localizes to the microtubule quartet, a cytoskeletal structure characteristic to trypanosomatids. Ablation of these proteins in Trypansoma brucei leads to distinct morphological defects, making them not only intrinsically interesting topics of study, but potential therapeutic targets as well.