Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.     

Regulation of G Protein-Coupled Receptor Signaling by A-Kinase Anchoring Proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

Protein Kinase A and Phosphodiesterase-4D3 Binding to Coding Polymorphisms of Cardiac Muscle Anchoring Protein (mAKAP)

Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of this study was to examine the effects of naturally occurring mutations in human mAKAP on PKA and PDE4D3 signaling. We have recently identified potentially important human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to β-adrenergic receptor signaling. Three mutations (P1400S, S2195F, and L717V) were cloned and transfected into a mammalian cell line for the purpose of comparing whether those substitutions disrupt mAKAP binding to PKA or PDE4D3. Immunoprecipitation study of mAKAP-P1400S, a mutation located in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding to PDE4D3, with no significant changes in PKA binding or PKA activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site, showed significant increase in both binding propensity to PKA and PKA activity. Additionally, mAKAP-L717V, a mutation flanking the mAKAP-spectrin repeat domain, exhibited a significant increase in PKA binding compared to wild type, but there was no change in PKA activity. We also demonstrate specific binding of wild-type mAKAP to PDE4D3. Binding results were demonstrated using immunoprecipitation and confirmed with surface plasmon resonance (Biacore-2000); functional results were demonstrated using activity assays, Ca^2 + measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling.     

Membrane anchoring γ-secretase modulators with terpene-derived moieties

Modulation of γ-secretase activity is a promising therapeutic strategy for the treatment of Alzheimer’s disease. Herein we report on the synthesis of carprofen- and tocopherol-derived small-molecule modulators carrying terpene moieties as lipophilic membrane anchors. Additionally, these modulators are equipped with an acidic moiety, which contributes to the desired modulatory effect on the γ-secretase with decreased formation of Aβ₄₂ and increased Aβ₃₈ production.     

α-Synemin localizes to the M-band of the sarcomere through interaction with the M10 region of titin

α-Synemin contains a unique 312 amino acid insert near the end of its C-terminal tail. Therefore we set out to determine if the insert is a site of protein–protein interaction that regulates the sub-cellular localization of this large isoform of synemin. Yeast-two hybrid analysis indicated that this region is a binding site for the M10 region of titin. This was confirmed with GST pull-down assays. Co-immunoprecipitation of endogenous proteins indicated close association of the two proteins in vivo and immunostaining of cardiomyocytes demonstrated co-localization of the proteins at the M-band of the sarcomere.     

A metabolomics approach to assessing phytotoxic effects on the green alga Scenedesmus vacuolatus

The potential of metabolomics for toxicity analysis with synchronized algal populations during growth was explored in a proof of principle study. Low molecular weight compounds from hydrophilic and lipophilic extracts of algal populations of the unicellular green alga Scenedesmus vacuolatus were analyzed using gas chromatography-mass spectrometry (GC-MS) and subsequent multivariate analysis to identify time-related patterns. Algal metabolite responses were studied under control and exposure conditions for the photosystem II-inhibiting herbicide prometryn. To define the typical metabolic profile of control S. vacuolatus cultures seven time points over a growth period of 14 h were evaluated. The results show a clear time-related trend in metabolite levels and a distinct separation of exposed and reference algal populations. The results suggest an impairment of the energy metabolism associated with an activation of catabolic processes and a retardation of carbohydrate biosynthesis in treated algae. Metabolite results were compared to observation parameters, currently used in phytotoxicity assessment, showing that metabolites respond faster to exposure than algal growth. The potential of metabolomics for toxicity evaluation, especially to identify physiological markers and to detect effects at an early state of exposure, are discussed. Therefore, we suggest a metabolomics approach utilizing synchronous algal cultures to be a suitable future tool in ecotoxicology.     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

MOLECULAR MECHANISMS IN DEVELOPMENTAL BIOLOGY

Some general molecular mechanisms underlying development are described. Namely: those involved in the differentiation of the R7 receptor inDrosophilaembryonic retina; those involved in the determination of embryonic axes and in polar cell differentiation, inDrosophila; those involved in the determination of the AB and P cell lineage and in vulva differentiation inCaenorhabditisembryos.     

丝状真菌糖生物学

蛋白质的糖基化修饰是一种保守的真核生物蛋白质翻译后修饰,存在于从酵母到人的所有真核生物中,赋予了蛋白质功能的多样性。目前对蛋白质糖基化修饰的了解主要来源于对酵母和哺乳动物细胞的研究,但单细胞真核生物或动物细胞水平的研究,很难反映糖基化修饰在多细胞真核生物的发育分化过程中的复杂功能。由于丝状真菌是多细胞真核生物,有相对简单的发育分化过程,因而是研究多细胞真核生物糖基化功能的理想模型之一,在过去10年中,丝状真菌的糖生物学研究开始受到重视,目前的研究结果表明糖基化修饰与丝状真菌的生长、发育密切相关。     

Protein kinase A‐mediated phosphorylation of naked cuticle homolog 2 stimulates cell‐surface delivery of transforming growth factor‐α for epidermal growth factor receptor transactivation

The classic mode of G protein‐coupled receptor (GPCR)‐mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)‐mediated cleavage of plasma membrane‐anchored EGFR ligands. Herein, we show that the Gαs‐activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E₂ (PGE₂) transactivate EGFR through increased cell‐surface delivery of the EGFR ligand transforming growth factor‐α (TGFα) in polarizing madin‐darby canine kidney (MDCK) and Caco‐2 cells. This is achieved by PKA‐mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα‐containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE₂ rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser‐223, a process that is facilitated by the molecular scaffold A‐kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell‐surface delivery of TGFα and increased EGFR activation. Thus, GPCR‐triggered, PKA/AKAP12/NKD2‐regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.