Phosphorylation at the disordered N-end makes HuR accumulate and dimerize in the cytoplasm.

Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.

Genetic variants affecting mitochondrial function provide further insights for kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a complex disorder that has become a high prevalence global health problem, with diabetes being its predominant pathophysiologic driver. Autosomal genetic variation only explains some of the predisposition to kidney disease. Variations in the mitochondrial genome (mtDNA) and nuclear-encoded mitochondrial genes (NEMG) are implicated in susceptibility to kidney disease and CKD progression, but they have not been thoroughly explored. Our aim was to investigate the association of variation in both mtDNA and NEMG with CKD (and related traits), with a particular focus on diabetes. METHODS: We used the UK Biobank (UKB) and UK-ROI, an independent collection of individuals with type 1 diabetes mellitus (T1DM) patients. RESULTS: Fourteen mitochondrial variants were associated with estimated glomerular filtration rate (eGFR) in UKB. Mitochondrial variants and haplogroups U, H and J were associated with eGFR and serum variables. Mitochondrial haplogroup H was associated with all the serum variables regardless of the presence of diabetes. Mitochondrial haplogroup X was associated with end-stage kidney disease (ESKD) in UKB. We confirmed the influence of several known NEMG on kidney disease and function and found novel associations for SLC39A13, CFL1, ACP2 or ATP5G1 with serum variables and kidney damage, and for SLC4A1, NUP210 and MYH14 with ESKD. The G allele of TBC1D32-rs113987180 was associated with higher risk of ESKD in patients with diabetes (OR:9.879; CI95%:4.440-21.980; P = 2.0E-08). In UK-ROI, AGXT2-rs71615838 and SURF1-rs183853102 were associated with diabetic nephropathies, and TFB1M-rs869120 with eGFR. CONCLUSIONS: We identified novel variants both in mtDNA and NEMG which may explain some of the missing heritability for CKD and kidney phenotypes. We confirmed the role of MT-ND5 and mitochondrial haplogroup H on renal disease (serum variables), and identified the MT-ND5-rs41535848G variant, along with mitochondrial haplogroup X, associated with higher risk of ESKD. Despite most of the associations were independent of diabetes, we also showed potential roles for NEMG in T1DM.

Mitochondrial related variants associated with cardiovascular traits.

Introduction: Cardiovascular disease (CVD) is responsible for over 30% of mortality worldwide. CVD arises from the complex influence of molecular, clinical, social, and environmental factors. Despite the growing number of autosomal genetic variants contributing to CVD, the cause of most CVDs is still unclear. Mitochondria are crucial in the pathophysiology, development and progression of CVDs; the impact of mitochondrial DNA (mtDNA) variants and mitochondrial haplogroups in the context of CVD has recently been highlighted. Aims: We investigated the role of genetic variants in both mtDNA and nuclear-encoded mitochondrial genes (NEMG) in CVD, including coronary artery disease (CAD), hypertension, and serum lipids in the UK Biobank, with sub-group analysis for diabetes. Methods: We investigated 371,542 variants in 2,527 NEMG, along with 192 variants in 32 mitochondrial genes in 381,994 participants of the UK Biobank, stratifying by presence of diabetes. Results: Mitochondrial variants showed associations with CVD, hypertension, and serum lipids. Mitochondrial haplogroup J was associated with CAD and serum lipids, whereas mitochondrial haplogroups T and U were associated with CVD. Among NEMG, variants within Nitric Oxide Synthase 3 (NOS3) showed associations with CVD, CAD, hypertension, as well as diastolic and systolic blood pressure. We also identified Translocase Of Outer Mitochondrial Membrane 40 (TOMM40) variants associated with CAD; Solute carrier family 22 member 2 (SLC22A2) variants associated with CAD and CVD; and HLA-DQA1 variants associated with hypertension. Variants within these three genes were also associated with serum lipids. Conclusion: Our study demonstrates the relevance of mitochondrial related variants in the context of CVD. We have linked mitochondrial haplogroup U to CVD, confirmed association of mitochondrial haplogroups J and T with CVD and proposed new markers of hypertension and serum lipids in the context of diabetes. We have also evidenced connections between the etiological pathways underlying CVDs, blood pressure and serum lipids, placing NOS3, SLC22A2, TOMM40 and HLA-DQA1 genes as common nexuses.

Hu Antigen R (HuR) Protein Structure, Function and Regulation in Hepatobiliary Tumors.

Hu antigen R (HuR) is a 36-kDa ubiquitous member of the ELAV/Hu family of RNA-binding proteins (RBPs), which plays an important role as a post-transcriptional regulator of specific RNAs under physiological and pathological conditions, including cancer. Herein, we review HuR protein structure, function, and its regulation, as well as its implications in the pathogenesis, progression, and treatment of hepatobiliary cancers. In particular, we focus on hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), tumors where the increased cytoplasmic localization of HuR and activity are proposed, as valuable diagnostic and prognostic markers. An overview of the main regulatory axes involving HuR, which are associated with cell proliferation, invasion, metastasis, apoptosis, and autophagy in HCC, is provided. These include the transcriptional, post-transcriptional, and post-translational modulators of HuR function, in addition to HuR target transcripts. Finally, whereas studies addressing the relevance of targeting HuR in CCA are limited, in the past few years, HuR has emerged as a potential therapeutic target in HCC. In fact, the therapeutic efficacy of some pharmacological inhibitors of HuR has been evaluated, in early experimental models of HCC. We, further, discuss the major findings and future perspectives of therapeutic approaches that specifically block HuR interactions, either with post-translational modifiers or cognate transcripts in hepatobiliary cancers.

Post-translational Control of RNA-Binding Proteins and Disease-Related Dysregulation.

Cell signaling mechanisms modulate gene expression in response to internal and external stimuli. Cellular adaptation requires a precise and coordinated regulation of the transcription and translation processes. The post-transcriptional control of mRNA metabolism is mediated by the so-called RNA-binding proteins (RBPs), which assemble with specific transcripts forming messenger ribonucleoprotein particles of highly dynamic composition. RBPs constitute a class of trans-acting regulatory proteins with affinity for certain consensus elements present in mRNA molecules. However, these regulators are subjected to post-translational modifications (PTMs) that constantly adjust their activity to maintain cell homeostasis. PTMs can dramatically change the subcellular localization, the binding affinity for RNA and protein partners, and the turnover rate of RBPs. Moreover, the ability of many RBPs to undergo phase transition and/or their recruitment to previously formed membrane-less organelles, such as stress granules, is also regulated by specific PTMs. Interestingly, the dysregulation of PTMs in RBPs has been associated with the pathophysiology of many different diseases. Abnormal PTM patterns can lead to the distortion of the physiological role of RBPs due to mislocalization, loss or gain of function, and/or accelerated or disrupted degradation. This Mini Review offers a broad overview of the post-translational regulation of selected RBPs and the involvement of their dysregulation in neurodegenerative disorders, cancer and other pathologies.

Exploring protein phosphorylation by combining computational approaches and biochemical methods.

Post-translational modifications of proteins expand their functional diversity, regulating the response of cells to a variety of stimuli. Among these modifications, phosphorylation is the most ubiquitous and plays a prominent role in cell signaling. The addition of a phosphate often affects the function of a protein by altering its structure and dynamics. However, these alterations are often difficult to study and the functional and structural implications remain unresolved. New approaches are emerging to overcome common obstacles related to the production and manipulation of these samples. Here, we summarize the available methods for phosphoprotein purification and phosphomimetic engineering, highlighting the advantages and disadvantages of each. We propose a general workflow for protein phosphorylation analysis combining computational and biochemical approaches, building on recent advances that enable user-friendly and easy-to-access Molecular Dynamics simulations. We hope this innovative workflow will inform the best experimental approach to explore such post-translational modifications. We have applied this workflow to two different human protein models: the hemeprotein cytochrome c and the RNA binding protein HuR. Our results illustrate the usefulness of Molecular Dynamics as a decision-making tool to design the most appropriate phosphomimetic variant.