S-acylation of the insulin-responsive aminopeptidase (IRAP) : quantitative analysis and identification of modified cysteines

The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation. In this study, we employ a semi-quantitative acyl-RAC technique to show that approximately 60% of IRAP is S-acylated in 3T3-L1 adipocytes. In contrast, S-acylation of GLUT4, a glucose transporter that extensively co-localises with IRAP, was approximately five-fold lower. Site-directed mutagenesis was employed to map the sites of S-acylation on IRAP to two cysteine residues, one of which is predicted to lie in the cytoplasmic side of the single transmembrane domain and the other which is just upstream of this transmembrane domain; our results suggest that these cysteines may be modified in a mutually-exclusive manner. Although S-acylation regulates the intracellular trafficking of several transmembrane proteins, we did not detect any effects of mutating the modified cysteines on the plasma membrane localisation of IRAP in HEK293T cells, suggesting that S-acylation is not essential for the movement of IRAP through the secretory pathway

The zDHHC family of S-acyltransferases

The discovery of the zDHHC family of S-acyltransferase enzymes has been one of the major breakthroughs in the S-acylation field. Now, more than a decade since their discovery, major questions centre on profiling the substrates of individual zDHHC enzymes (there are 24 ZDHHC genes and several hundred S-acylated proteins), defining the mechanisms of enzyme-substrate specificity and unravelling the importance of this enzyme family for cellular physiology and pathology

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Analysis of protein palmitoylation in adipocytes

Blood glucose homeostasis is highly regulated and is essential for survival. A key process is the insulin-stimulated recruitment of the facilitative glucose transporter GLUT4 to the plasma membrane in adipocytes and muscle cells; defects in this pathway can cause insulin resistance and type 2 diabetes. The insulin signalling and GLUT4 trafficking pathways in these cells have been extensively characterised, and a prominent role for protein phosphorylation has been uncovered. In contrast, relatively little is known about the role of other post-translational modifications (PTMs) in these pathways. The aim of this study was to expand existing knowledge of how palmitoylation, a PTM involving reversible attachment of fatty acids onto cysteine residues, affects components of the insulin signalling and GLUT4 trafficking pathways. For this, palmitoylated proteins were isolated from 3T3-L1 adipocytes by resin-assisted capture of S-acylated proteins (acyl-RAC), and screened to identify novel palmitoylated components of the insulin signalling and GLUT4 trafficking pathways. This approach successfully identified the following novel palmitoylated proteins: GLUT4, insulin-responsive aminopeptidase (IRAP) and caveolin-2. Furthermore, click-chemistry confirmed palmitoylation of caveolin-2 and IRAP and also enabled identification of the palmitoylation sites on these proteins. Palmitoylation has been shown to regulate proteins in many different ways, in particular by modulating protein trafficking, protein stability and protein-protein interactions. Mutation of the palmitoylation sites in caveolin-2 and IRAP had no obvious effect on protein localisation. However, palmitoylation-deficient mutants of caveolin-2 exhibited: (i) a deficit in conversion of monomeric caveolin-2 into low molecular weight oligomeric complexes, and (ii) decreased oligomer stability, revealed by a loss of SDS-resistant caveolin-2 complexes. Overall, this work has identified novel palmitoylated proteins in 3T3-L1 adipocytes, mapped the palmitoylation sites of these proteins, and determined the effect of this PTM on the localisation and oligomeric status of these proteins. These findings have thus expanded existing knowledge on the potential regulation of insulin signalling and GLUT4 trafficking pathways by PTMs.Blood glucose homeostasis is highly regulated and is essential for survival. A key process is the insulin-stimulated recruitment of the facilitative glucose transporter GLUT4 to the plasma membrane in adipocytes and muscle cells; defects in this pathway can cause insulin resistance and type 2 diabetes. The insulin signalling and GLUT4 trafficking pathways in these cells have been extensively characterised, and a prominent role for protein phosphorylation has been uncovered. In contrast, relatively little is known about the role of other post-translational modifications (PTMs) in these pathways. The aim of this study was to expand existing knowledge of how palmitoylation, a PTM involving reversible attachment of fatty acids onto cysteine residues, affects components of the insulin signalling and GLUT4 trafficking pathways. For this, palmitoylated proteins were isolated from 3T3-L1 adipocytes by resin-assisted capture of S-acylated proteins (acyl-RAC), and screened to identify novel palmitoylated components of the insulin signalling and GLUT4 trafficking pathways. This approach successfully identified the following novel palmitoylated proteins: GLUT4, insulin-responsive aminopeptidase (IRAP) and caveolin-2. Furthermore, click-chemistry confirmed palmitoylation of caveolin-2 and IRAP and also enabled identification of the palmitoylation sites on these proteins. Palmitoylation has been shown to regulate proteins in many different ways, in particular by modulating protein trafficking, protein stability and protein-protein interactions. Mutation of the palmitoylation sites in caveolin-2 and IRAP had no obvious effect on protein localisation. However, palmitoylation-deficient mutants of caveolin-2 exhibited: (i) a deficit in conversion of monomeric caveolin-2 into low molecular weight oligomeric complexes, and (ii) decreased oligomer stability, revealed by a loss of SDS-resistant caveolin-2 complexes. Overall, this work has identified novel palmitoylated proteins in 3T3-L1 adipocytes, mapped the palmitoylation sites of these proteins, and determined the effect of this PTM on the localisation and oligomeric status of these proteins. These findings have thus expanded existing knowledge on the potential regulation of insulin signalling and GLUT4 trafficking pathways by PTMs

Palmitoylation and the trafficking of peripheral membrane proteins

Palmitoylation, the attachment of palmitate and other fatty acids on to cysteine residues, is a common post-translational modification of both integral and peripheral membrane proteins. Dynamic palmitoylation controls the intracellular distribution of peripheral membrane proteins by regulating membrane-cytosol exchange and/or by modifying the flux of the proteins through vesicular transport systems

The management of acute venous thromboembolism in clinical practice - study rationale and protocol of the European PREFER in VTE Registry

Background: Venous thromboembolism (VTE) is a major health problem, with over one million events every year in Europe. However, there is a paucity of data on the current management in real life, including factors influencing treatment pathways, patient satisfaction, quality of life (QoL), and utilization of health care resources and the corresponding costs. The PREFER in VTE registry has been designed to address this and to understand medical care and needs as well as potential gaps for improvement. Methods/design: The PREFER in VTE registry was a prospective, observational, multicenter study conducted in seven European countries including Austria, France Germany, Italy, Spain, Switzerland, and the UK to assess the characteristics and the management of patients with VTE, the use of health care resources, and to provide data to estimate the costs for 12 months treatment following a first-time and/or recurrent VTE diagnosed in hospitals or specialized or primary care centers. In addition, existing anticoagulant treatment patterns, patient pathways, clinical outcomes, treatment satisfaction, and health related QoL were documented. The centers were chosen to reflect the care environment in which patients with VTE are managed in each of the participating countries. Patients were eligible to be enrolled into the registry if they were at least 18 years old, had a symptomatic, objectively confirmed first time or recurrent acute VTE defined as either distal or proximal deep vein thrombosis, pulmonary embolism or both. After the baseline visit at the time of the acute VTE event, further follow-up documentations occurred at 1, 3, 6 and 12 months. Follow-up data was collected by either routinely scheduled visits or by telephone calls. Results: Overall, 381 centers participated, which enrolled 3,545 patients during an observational period of 1 year. Conclusion: The PREFER in VTE registry will provide valuable insights into the characteristics of patients with VTE and their acute and mid-term management, as well as into drug utilization and the use of health care resources in acute first-time and/or recurrent VTE across Europe in clinical practice. Trial registration: Registered in DRKS register, ID number: DRKS0000479