PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond?

Dysregulated signal transduction in innate and adaptive immune cells is known to be associated with the development of various autoimmune and inflammatory diseases. Consequently, targeting intracellular signalling of the pro-inflammatory cytokine network heralds hope for the next generation of anti-inflammatory drugs. Phosphoinositide 3-kinases (PI3Ks) generate lipid-based second messengers that control an array of intracellular signalling pathways that are known to have important roles in leukocytes. In light of the recent progress in the development of selective PI3K inhibitors, and the beneficial effects of these inhibitors in models of acute and chronic inflammatory disorders, we discuss the therapeutic potential of blocking PI3K isoforms for the treatment of rheumatoid arthritis and other immune-mediated diseases.     

Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.     

Kaposi’s Sarcoma-Associated Herpesvirus K3 and K5 Proteins Down Regulate Both DC-SIGN and DC-SIGNR

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of multicentric Castleman’s disease, primary effusion lymphoma and Kaposi’s sarcoma. In this study, we show that like the C-type lectin DC-SIGN, the closely related DC-SIGNR can also enhance KSHV infection. Following infection, they are both targeted for down modulation and our data indicate that the KSHV MARCH-family ubiquitin ligase K5 is mediating this regulation and subsequent targeting for degradation of DC-SIGN and DC-SIGNR in the context of the virus. The closely related viral K3 protein, is also able to target these lectins in exogenous expressions studies, but only weakly during viral infection. In addition to requiring a functional RING-CH domain, several protein trafficking motifs in the C-terminal region of both K3 and K5 are important in regulation of DC-SIGN and DC-SIGNR. Further exploration of this modulation revealed that DC-SIGN is endocytosed from the cell surface in THP-1 monocytes, but degraded from an internal location with minimal endocytosis in HEK-293 cells. Pull-down data indicate that both K3 and K5 preferentially associate with immature forms of the lectins, mediating their ubiquitylation and degradation. Together, these data emphasize the molecular complexities of K3 and K5, while expanding the repertoire of targets of these two viral proteins.     

Insulin-Driven PI3K-AKT Signaling in the Hepatocyte Is Mediated by Redundant PI3Kα and PI3Kβ Activities and Is Promoted by RAS

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CoA Synthase is in complex with p85αPI3K and affects PI3K signaling pathway

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA Synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85αPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85αPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110α and regulatory p85α subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85αPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.     

PI3K and PKC contribute to membrane depolarization mediated by α<Subscript>2</Subscript>-adrenoceptors in the canine isolated mesenteric vein

Background  

Norepinephrine (NE), a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are involved in the electrical field stimulation (EFS)-induced slow membrane depolarization (SMD) in canine isolated mesenteric vein. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s)-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3) receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined.     

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.     

Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant E.coli

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level.In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins.Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control.The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.     

泛素连接酶E3

蛋白质的泛素化修饰具有高度的特异性,它参与调节细胞内许多的生理活动。蛋白质的泛素化修饰涉及一系列的酶参与反应,包括泛素激活酶E1、结合酶E2以及连接酶E3。而其中泛素连接酶E3对靶蛋白的特异性识别起关键作用。泛素连接酶E3主要由HECT结构域家族、RING结构域家族和U-box结构域家族组成。现对泛素连接酶E3的分类、结构及其对靶蛋白的识别机制等进行综述。     

Phosphoinositide 3-kinase delta (PI3Kδ) in leukocyte signaling and function

PI3Kδ is a lipid kinase of the PI3K class IA family involved in early signaling events of leukocytes responding to a wide variety of stimuli. The leukocyte specificity of PI3Kδ is defined by its expression, whereas its signaling function is via the production of phosphoinositide 3,4,5-triphosphates at the proximity of activated receptors for recruiting other signaling molecules. The importance of PI3Kδ in B cell development and function is most apparent, and its role in other leukocyte cell types can be easily demonstrated as well. PI3Kδ participates in the development, activation and migration of T cells and NK cells. The role of PI3Kδ in myeloid cell activities, such as inflammation driven cell infiltration, neutrophil oxidative burst, immune complex mediated macrophage activation, as well as mast cell maturation and degranulation, has been well illustrated in various studies. As a result of the broad effects of PI3Kδ in leukocyte functions, the disruption of PI3Kδ expression or activity leads to decreased inflammatory and immune responses in vivo. The protective role of PI3Kδ inactivation in animal models of arthritis, asthma or obstructive respiratory diseases has been demonstrated. These findings suggest the potential efficacy achievable with PI3Kδ inhibitors in the treatment of autoimmune and respiratory diseases.     

A gene for speed? The evolution and function of α‐actinin‐3

The alpha-actinins are an ancient family of actin-binding proteins that play structural and regulatory roles in cytoskeletal organisation and muscle contraction. alpha-actinin-3 is the most-highly specialised of the four mammalian alpha-actinins, with its expression restricted largely to fast glycolytic fibres in skeletal muscle. Intriguingly, a significant proportion ( approximately 18%) of the human population is totally deficient in alpha-actinin-3 due to homozygosity for a premature stop codon polymorphism (R577X) in the ACTN3 gene. Recent work in our laboratory has revealed a strong association between R577X genotype and performance in a variety of athletic endeavours. We are currently exploring the function and evolutionary history of the ACTN3 gene and other alpha-actinin family members. The alpha-actinin family provides a fascinating case study in molecular evolution, illustrating phenomena such as functional redundancy in duplicate genes, the evolution of protein function, and the action of natural selection during recent human evolution.     

14-3-3γ Induces Oncogenic Transformation by Stimulating MAP Kinase and PI3K Signaling

The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3γ was shown by focus formation and tumor formation in SCID mice using 14-3-3γ transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3σ, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3γ. In addition, we found that 14-3-3γ interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3γ as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3γ induced transformation.     

Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation

Human cystathionine β-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H₂S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by ∼28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that γ-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.     

K2000E发动机燃油系统冷却改造

通过对MT-3600B矿用汽车K2000E发动机燃油泵系统工艺探讨,结合实际工作中不断出现的高怠速问题,总结分析系统故障出现的原因,从而有针对性地改变怠速不稳及高怠等现象,降低运行成本,使矿用汽车能安全可靠的平稳运行。     

Calcium oscillation and phosphatidylinositol 3-kinase positively regulate integrin α<Subscript>IIb</Subscript>β<Subscript>3</Subscript>-mediated outside-in signaling

Summary The frequency of calcium oscillation reveals the platelet activation status, however, the biological significance of the periodic calcium responses and methods of communication with other integrin-mediated signals are not clear. RGD-containing disintegrin rhodostomin coated substrates were employed to enhance platelet spreading and calcium oscillation through direct binding and clustering of the receptor integrin _IIb3. The results showed that the activation of phosphatidylinositol 3-kinase (PI3-K) and internal calcium pathways were crucial for _IIb3 outside-in signaling. PI3-K antagonists wortmannin and LY294002 inhibited disintegrin substrates and induced platelet spreading and calcium oscillation. At the same time, pretreatment of platelets with the microsomal calcium–ATPase inhibitor thapsigargin to deplete internal calcium stores severely impaired the calcium oscillation as well as PI3-K activation and spreading on disintegrin substrates. Because inhibition of one pathway could inhibit the other, our data indicates that PI3-K and calcium oscillation are synergistically operated and form a positive-feedback regulation in integrin _IIb3-mediated outside-in signaling.     

Calmodulin and IQGAP1 activation of PI3Kα and Akt in KRAS,HRAS and NRAS-driven cancers

Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP₃. CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS- and NRAS-driven cancers.