Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry

To investigate protein–protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH–MutL–DNA complex

Spatial variability in herbaceous plant phenology is mostly explained by variability in temperature but also by photoperiod and functional traits

Whereas temporal variability of plant phenology in response to climate change has already been well studied, the spatial variability of phenology is not well understood. Given that phenological shifts may affect biotic interactions, there is a need to investigate how the variability in environmental factors relates to the spatial variability in herbaceous species’ phenology by at the same time considering their functional traits to predict their general and species-specific responses to future climate change. In this project, we analysed phenology records of 148 herbaceous species, which were observed for a single year by the PhenObs network in 15 botanical gardens. For each species, we characterised the spatial variability in six different phenological stages across gardens. We used boosted regression trees to link these variabilities in phenology to the variability in environmental parameters (temperature, latitude and local habitat conditions) as well as species traits (seed mass, vegetative height, specific leaf area and temporal niche) hypothesised to be related to phenology variability. We found that spatial variability in the phenology of herbaceous species was mainly driven by the variability in temperature but also photoperiod was an important driving factor for some phenological stages. In addition, we found that early-flowering and less competitive species characterised by small specific leaf area and vegetative height were more variable in their phenology. Our findings contribute to the field of phenology by showing that besides temperature, photoperiod and functional traits are important to be included when spatial variability of herbaceous species is investigated

The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells

Lipidomics needs more standardization

Modern mass spectrometric technologies provide quantitative readouts for a wide variety of lipid specimens. However, many studies do not report absolute lipid concentrations and differ vastly in methodologies, workflows, and data presentation. Therefore, we appeal to researchers to engage with the Lipidomics Standards Initiative to develop common standards for minimum acceptable data quality and reporting for lipidomics data to take lipidomics research to the next level

Quantification of bulk lipid species in human platelets and their thrombin-induced release

Lipids play a central role in platelet physiology. Changes in the lipidome have already been described for basal and activated platelets. However, quantitative lipidomic data of platelet activation, including the released complex lipids, are unavailable. Here we describe an easy-to-use protocol based on flow-injection mass spectrometry for the quantitative analysis of bulk lipid species in basal and activated human platelets and their lipid release after thrombin activation. We provide lipid species concentrations of 12 healthy human donors, including cholesteryl ester (CE), ceramide (Cer), free cholesterol (FC), hexosylceramide (HexCer), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM) and triglycerides (TG). The assay exhibited good technical repeatability (CVs < 5% for major lipid species in platelets). Except for CE and TG, the inter-donor variability of the majority of lipid species concentrations in platelets was < 30% CV. Balancing of concentrations revealed the generation of LPC and loss of TG. Changes in lipid species concentrations indicate phospholipase-mediated release of arachidonic acid mainly from PC, PI, and PE but not from PS. Thrombin induced lipid release was mainly composed of FC, PS, PC, LPC, CE, and TG. The similarity of the released lipidome with that of plasma implicates that lipid release may originate from the open-canalicular system (OCS). The repository of lipid species concentrations determined with this standardized platelet release assay contribute to elucidating the physiological role of platelet lipids and provide a basis for investigating the platelet lipidome in patients with hemorrhagic or thrombotic disorders

Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome.

Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe-/- and Ldlr-/- mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe-/- mice). Bone marrow from wild-type or Ldlr-/- mice was transplanted into irradiated Ldlr-/- recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe-/- and Ldlr-/- mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype

Coactosin-like 1 integrates signaling critical for shear-dependent thrombus formation in mouse platelets

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis

Quality control requirements for the correct annotation of lipidomics data.

10.1038/s41467-021-24984-yNature Communications121477