o-Nitrotyrosine and p-iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes

High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clear-cut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner.     

Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson’s diseases

Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging‐related diseases, in a mouse model of PD induced by 1‐methyl‐4‐phenyl‐1,2,4,6‐tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP‐induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α‐synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α‐synuclein oligomerization and S‐129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.     

Protein biomarkers for response to XPO1 inhibition in haematologic malignancies

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.     

Palmitic acid induces IP-10 expression in human macrophages via NF-kappaB activation

It is now recognized that cross-talk between adipocytes and adipose tissue stromal cells such as macrophages contributes to local and systemic inflammation. One factor from adipocytes that may participate in this interaction and that is frequently elevated in inflammatory conditions such as obesity, insulin resistance, and type 2 diabetes is free fatty acids (FFA). To investigate the potential for FFA to enhance macrophage inflammation, we exposed U937 macrophages to physiological levels (150 microM) of FFA. Palmitic acid (PA), the predominant saturated FFA released from adipose tissue, but not unsaturated FFA, induced an approximately 6-fold (p<0.05) increase in IP-10 gene expression (and 2- to 4-fold increases in IL-8, MCP-1, COX-2, and MIG). PA also induced an approximately 2-fold increase (p<0.05) in active NF-kappaB, and two structurally distinct NF-kappaB inhibitors effectively blocked PA-induced IP-10 gene expression. Conditioned medium from PA-treated cells increased lymphocyte migration 41% (p<0.05) which was significantly reduced by IP-10-neutralizing antibody. These results suggest that elevated concentrations of PA commonly present in obese and insulin resistant individuals can increase NF-kappaB-mediated expression of IP-10 in macrophages. These events in turn may lead to an increasing feed-forward loop of chronic inflammation.     

Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli beta-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65 degrees C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C of N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.     

POPDC1 scaffolds a complex of adenylyl cyclase 9 and the potassium channel TREK‐1 in heart

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain‐containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP‐dependent binding and regulation of TREK‐1 potassium channels. We show that TREK‐1 binds the AC9:POPDC1 complex and copurifies in a POPDC1‐dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP‐independent, TREK‐1 association with AC9 and POPDC1 is reduced upon stimulation of the β‐adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK‐1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK‐1 currents. Finally, deletion of the gene‐encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress‐induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK‐1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK‐1 to regulate heart rate control.     

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.