Sidney W. Whiteheart

AAA+ proteins: have engine, will work.

The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The familys defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.

PKCα regulates platelet granule secretion and thrombus formation in mice

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA

Summary.  Background: Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. Objectives: To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. Subjects/methods: Two hundred and eighty‐eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n = 18) and hyporeactivity (n = 11) was used to screen the human transcriptome. Results: Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v‐SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q = 0.0275). Validation studies of microarray results showed 4.8‐fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P = 0.0023). VAMP8 protein levels varied 13‐fold among platelets from these normal subjects, and were 2.5‐fold higher in hyperreactive platelets (P = 0.05). Among our cohort of 288 subjects, a VAMP8 single‐nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age‐dependent manner (P < 0.003). MicroRNA‐96 was predicted to bind to the 3′‐untranslated regionof VAMP8 mRNA and was detected in platelets. Overexpression of microRNA‐96 in VAMP8‐expressing cell lines caused a dose‐dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. Conclusions: These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA‐96 in the regulation of VAMP8 expression.

TLR Signals Induce Phagosomal MHC-I Delivery from the Endosomal Recycling Compartment to Allow Cross-Presentation

Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection.

N-ethylmaleimide sensitive factor (NSF) structure and function.

Our understanding of the molecular mechanisms of membrane trafficking advanced at a rapid rate during the 1990s. As one of the initial protein components of the trafficking machinery to be identified, N-ethylmaleimide sensitive factor (NSF) has served as a reference point in many of these recent studies. This hexameric ATPase is essential for most of the membrane-trafficking events in a cell. Initially, due to its ATPase activity, NSF was thought to be the motor that drove membrane fusion. Subsequent studies have shown that NSF actually plays the role of a chaperone by activating SNAP receptor proteins (SNAREs) so that they can participate in membrane fusion. In this review we will examine the initial characterization of NSF, its role in membrane fusion events, and what new structural information can tell us about NSFs mechanism of action.

Platelet secretion is kinetically heterogeneous in an agonist-responsive manner

Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically controlled process? Here we sought to describe the platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (ie, thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules, whereas thrombin rapidly induced the greatest release. Cargo with opposing actions (eg, proangiogenic and antiangiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with 3 classes of release events, differing in their rates. The distribution of cargo into these 3 classes was heterogeneous, suggesting that platelet secretion is a stochastic process potentially controlled by several factors, such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes.

Phosphorylation of SNAP-23 Regulates Exocytosis from Mast Cells

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser95 and Ser120 as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that ∼10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser95 and Ser120 is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser120 and Ser95 modulates regulated exocytosis by mast cells.

Crystal structure of the amino-terminal domain of N -ethylmaleimide-sensitive fusion protein

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein α-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.

Cellular functions of NSF: Not just SNAPs and SNAREs

N‐ethylmaleimide sensitive factor (NSF) is an ATPases associated with various cellular activities protein (AAA), broadly required for intracellular membrane fusion. NSF functions as a SNAP receptor (SNARE) chaperone which binds, through soluble NSF attachment proteins (SNAPs), to SNARE complexes and utilizes the energy of ATP hydrolysis to disassemble them thus facilitating SNARE recycling. While this is a major function of NSF, it does seem to interact with other proteins, such as the AMPA receptor subunit, GluR2, and β2‐AR and is thought to affect their trafficking patterns. New data suggest that NSF may be regulated by transient post‐translational modifications such as phosphorylation and nitrosylation. These new aspects of NSF function as well as its role in SNARE complex dynamics will be discussed.

The Platelet Release Reaction: Just when you thought platelet secretion was simple

Purpose of reviewIn response to agonists produced at vascular lesions, platelets release a host of components from their three granules: dense core, alpha, and lysosome. This releasate activates other platelets, promotes wound repair, and initiates inflammatory responses. Although widely accepted, the specific mechanisms underlying platelet secretion are only now coming to light. This review focuses on the core machinery required for platelet secretion. Recent findingsProteomic analyses have provided a catalog of the components released from activated platelets. Experiments using a combination of in-vitro secretion assays and knockout mice have led to assignments of both vesicle-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (v-SNARE) and target membrane SNARE to each of the three secretion events. SNARE knockout mice are also proving to be useful models for probing the role of platelet exocytosis in vivo. Other studies are beginning to identify SNARE regulators, which control when and where SNAREs interact during platelet activation. SummaryA complex set of protein–protein interactions control the membrane fusion events required for the platelet release reaction. SNARE proteins are the core elements but the proteins that control SNARE interactions represent key points at which platelet signaling cascades could affect secretion and thrombosis.