Biochemical Journal最新文献

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown. Combining biophysical measurements, functional assays, and computational modeling in the context of the model protein Yop1, we found that missense mutations have variable effects on DP1 dimer structure and in vitro tubulation activity, and provide mechanistic insights into the role of DP1 oligomerisation on membrane curvature stabilization. Whereas the mutations P71L and S75F decreased dimer homogeneity and led to polydisperse oligomerization and impaired membrane curving activity, A72E introduced new polar interactions between subunits that stabilized the Yop1 dimer and allowed robust tubule formation but prevented formation of more highly-curved lipoprotein particles (LPP). The introduction of a BRIL domain to the cytoplasmic loop of A72E rescued LPP formation, consistent with a requirement for dimer splaying in highly curved membranes. These results suggest that the membrane curving activity of DP1 proteins requires both dimer stability and conformational plasticity at the intermolecular interface.

Adaptor proteins play central roles in the assembly of molecular complexes and co-ordinated activation of specific pathways. Through their modular domain structure, the NCK family of adaptor proteins (NCK1 and NCK2) link protein targets via their single SRC Homology (SH) 2 and three SH3 domains. Classically, their SH2 domain binds to phosphotyrosine motif-containing receptors (e.g. receptor tyrosine kinases), while their SH3 domains bind polyproline motif-containing cytoplasmic effectors. Due to these functions being established for both NCK1 and NCK2, their roles were inaccurately assumed to be redundant. However, in contrast with this previously held view, NCK1 and NCK2 now have a growing list of paralog-specific functions, which underscores the need to further explore their differences. Here we review current evidence detailing how these two paralogs are unique, including differences in their gene/protein regulation, binding partners and overall contributions to cellular functions. To help explain these contrasting characteristics, we then discuss SH2/SH3 structural features, disordered interdomain linker regions and post-translational modifications. Together, this review seeks to highlight the importance of distinguishing NCK1 and NCK2 in research and to pave the way for investigations into the origins of their interaction specificity.

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

Hydrogen peroxide (H2O2) transport by aquaporins (AQP) is a critical feature for cellular redox signaling. However, the H2O2 permeation mechanism through these channels remains poorly understood. Through functional assays, two Plasma membrane Intrinsic Protein (PIP) AQP from Medicago truncatula, MtPIP2;2 and MtPIP2;3 have been identified as pH-gated channels capable of facilitating the permeation of both water (H2O) and H2O2. Employing a combination of unbiased and enhanced sampling molecular dynamics simulations, we investigated the key barriers and translocation mechanisms governing H2O2 permeation through these AQP in both open and closed conformational states. Our findings reveal that both H2O and H2O2 encounter their primary permeation barrier within the selectivity filter (SF) region of MtPIP2;3. In addition to the SF barrier, a second energetic barrier at the NPA (asparagine-proline-alanine) region that is more restrictive for the passage of H2O2 than for H2O, was found. This behavior can be attributed to a dissimilar geometric arrangement and hydrogen bonding profile between both molecules in this area. Collectively, these findings suggest mechanistic heterogeneity in H2O and H2O2 permeation through PIPs.

Elevated plasma levels of lipoprotein(a) (Lp(a)) are a prevalent, independent, and causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve disease. Lp(a) consists of a lipoprotein particle resembling low density lipoprotein and the covalently-attached glycoprotein apolipoprotein(a) (apo(a)). Novel therapeutics that specifically and potently lower Lp(a) levels are currently in advanced stages of clinical development, including in large, phase 3 cardiovascular outcomes trials. However, fundamental unanswered questions remain concerning some key aspects of Lp(a) biosynthesis and catabolism as well as the true pathogenic mechanisms of the particle. In this review, we describe the salient biochemical features of Lp(a) and apo(a) and how they underlie the disease-causing potential of Lp(a), the factors that determine plasma Lp(a) concentrations, and the mechanism of action of Lp(a)-lowering drugs.