John E. Burke

Phospholipase A2 Biochemistry

The phospholipase A2 (PLA2) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. There are five main types of PLA2: the secreted sPLA2’s, the cytosolic cPLA2’s, the Ca2+independent iPLA2’s, the PAF acetylhydrolases, and the lysosomal PLA2’s. This review focuses on the superfamily of PLA2 enzymes, and then uses three specific examples of these enzymes to examine the differing biochemistry of the three main types of these enzymes. These three examples are the GIA cobra venom PLA2, the GIVA cytosolic cPLA2, and the GVIA Ca2+-independent iPLA2.

Structural Basis for Activation and Inhibition of Class I Phosphoinositide 3-Kinases

The regulatory interactions between PI3Ks and their binding partners could be exploited for therapies. Phosphoinositide 3-kinases (PI3Ks) phosphorylate a hydroxyl group on phosphoinositide lipids. The 3-phosphorylated inositol lipids act as membrane-resident second messengers, recruiting downstream signaling components that control cell growth, proliferation, differentiation, survival, and motility. The best studied of the PI3Ks, the class I enzymes, are heterodimers with a catalytic and a regulatory subunit and have been implicated in many human diseases. Class I PI3Ks can be stimulated downstream of receptor tyrosine kinases and heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors as well as small G proteins of the Ras superfamily. Both the catalytic and regulatory subunits have a multidomain organization. Crystal structures, biochemical analysis, and oncogenic mutations in PI3Ks have shown that interdomain interactions are not static but undergo regulated conformational cycles, resulting in enzyme activation or inhibition. This Review, which contains 7 figures and 104 references, highlights the molecular details of how their regulatory partners selectively inhibit and activate PI3K isoforms. Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate–dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)—the common precursor of the different series of glycosphingolipids—that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer—vesicular and non-vesicular—through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes.

Opening up Vps34 protein complexes During intracellular membrane trafficking, large protein complexes regulate and adapt the activity of signal transducer enzymes such as the class III phosphatidylinositol 3-kinase Vps34. These large enzyme complexes are present in all eukaryotic cells, having widespread importance in neurodegeneration, aging, and cancer; however, a structural understanding has been lacking. Rostislavleva et al. provide atomic-resolution insights into the structures of the Vps34-containing protein complexes required for autophagy, endocytic sorting, and cytokinesis. The V-shaped complexes can undergo opening motions, which allows them to adapt to and phosphorylate membranes. Science, this issue p. 10.1126/science.aac7365 An atomic-resolution analysis provides insight into protein complexes required for autophagy, endocytic sorting, and cytokinesis. INTRODUCTION The lipid kinase Vps34/PIK3C3 phosphorylates phosphatidylinositol to yield phosphatidylinositol 3-phosphate (PI3P). Vps34 is important for processes that sort cargo to lysosomes, including phagocytosis, endocytic traffic, autophagy, and cytosol-to-vacuole transport. In mammalian cells, the enzyme also has roles in cytokinesis, signaling, recycling, and lysosomal tubulation. Vps34 is present in multiple complexes. Complex I functions in autophagy and contains Vps34, Vps15 (p150/PIK3R4 in mammals), Vps30/Atg6 (Beclin 1), and Atg14 (ATG14L). Complex II takes part in endocytic sorting (as well as autophagy and cytokinesis in mammalian cells) and contains the same subunits as complex I, except that it has Vps38 (UVRAG) instead of Atg14. These complexes are differentially regulated in stress responses. In autophagy, PI3P emerges on small tubular or vesicular structures associated with nascent autophagosomes. RATIONALE One of the most compelling questions is how the Vps34-containing complexes are organized and to what extent their intrinsic properties contribute to their differential activities in cells. To understand the mechanisms by which these complexes impart differential activities to Vps34, we sought to determine the structure of complex II and to characterize activities of Vps34 complexes on small and large vesicles. Because the complex resisted crystallization attempts, we screened 15 different nanobodies against the complex, and one of them enabled crystallization. RESULTS We obtained a 4.4 Å crystal structure of yeast complex II. The structure has a Y-shaped organization with the Vps15 and Vps34 subunits intertwining in one arm so that the Vps15 kinase domain interacts with the lipid-binding region of the Vps34 kinase domain. The other arm has a parallel Vps30/Vps38 heterodimer. This indicates that the complex might assemble by Vps15/Vps34 associating with Vps30/Vps38. This assembly path is consistent with in vitro reconstitution of complex II and suggests how the abundance of various Vps34-containing complexes might be dynamically controlled. The Vps34 C2 domain is the keystone to the organization of the complexes, and several structural elaborations of the domain that facilitate its interaction with all complex II subunits are essential to the cellular role of Vps34. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify localized changes in all four complex II subunits upon membrane binding. We identified a loop in Vps30 (referred to as the “aromatic finger”) that interacts directly with lipid membranes. Our assays showed that complexes I and II had similar activities on small vesicles (100 nm). In contrast, only complex II was active on giant unilamellar vesicles (GUVs) (2 to 20 μm). This activity was completely abolished by mutation of the aromatic finger. CONCLUSION The structure, HDX-MS, and functional data allowed us to devise a model of how Vps34 complexes adapt to membranes. The tips of both arms of complex II work together on membranes. The Vps30 aromatic finger in one arm is important for the efficient catalytic activity of the other arm. The conformational changes that we detected may allow the arms to open to accommodate low-curvature membranes such as GUVs and endosomes. Most of the interactions observed in the complex II structure are likely to be detected in complex I as well. The restriction of complex I activity in autophagy to membrane structures smaller than 100 nm may be related to the inactivity of complex I on GUVs in vitro. Structure of complex II and its activity on GUVs. In the Y-shaped complex II, the Vps30/Vps38 pair in one arm brackets the Vps15/Vps34 pair in the other arm. Tips of both arms bind membranes. Only wild-type complex II forms PI3P on GUVs; in contrast, complex I and the complex II aromatic finger mutant are inactive. PI3P is detected by a sensor protein (red) binding to GUVs (green). Both complexes I and II have similar activities on small vesicles. Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.

Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors

How to recruit membrane trafficking machinery PI4KIIIβ is a lipid kinase that underlies Golgi function and is enlisted in biological responses that require rapid delivery of membrane vesicles, such as during the extensive membrane remodeling that occurs at the end of cell division. Burke et al. determined the structure of PI4KIIIβ in a complex with the membrane trafficking GTPase Rab11a. The way in which the proteins interact gives PI4KIIIβ the ability to simultaneously recruit Rab11a and its effectors on specific membranes. Science, this issue p. 1035 A lipid kinase interacts with target membranes, a membrane trafficking guanosine triphosphatase, and its effectors simultaneously. Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)–containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases, and their effectors is unknown. Here, we describe structures of PI4Kβ (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is distinct compared with known structures of Rab complexes and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria.

Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.

The Group VIA-2 Ca2+-independent phospholipase A2 (GVIA-2 iPLA2) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA2 in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA2 as well as the interfacial lipid binding region.

Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective.

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).

Significance Pathology of many diseases depends on signaling by phosphoinositide 3-kinase gamma (PI3Kγ), the lipid kinase that is exquisitely adapted to activation downstream of heterotrimeric G-protein–coupled receptors (GPCRs). Using hydrogen–deuterium exchange mass spectrometry, we demonstrate the mechanism by which the p110γ catalytic subunit and its p101 regulatory subunit interact with G-protein Gβγ heterodimers liberated upon GPCR activation. We identify residues in both p110γ and p101 interacting with Gβγ heterodimers on membranes. This enabled us to generate Gβγ-insensitive p110γ and p101 variants that eliminate activation of PI3Kγ by Gβγs without affecting the enzyme’s basal activity or its activation by the small G-protein Ras. Ablating the interaction of PI3Kγ with Gβγ heterodimers attenuates signaling, chemotaxis, and transformation driven by a GPCR agonist in cell lines. Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein–coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme’s activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gβγ heterodimers, we mapped these regulatory interactions using hydrogen–deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gβγ heterodimers, leading to PI3Kγ activation. Mutating Gβγ-interaction sites of either p110γ or p101 ablates G-protein–coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen–deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gβγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gβγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gβγ heterodimers; and the β-adrenergic receptor kinase.

Synergy in activating class I PI3Ks

The class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that transduce a host of cellular signals and regulate a broad range of essential functions including growth, proliferation, and migration. As such, PI3Ks have pivotal roles in diseases such as cancer, diabetes, primary immune disorders, and inflammation. These enzymes are activated downstream of numerous activating stimuli including receptor tyrosine kinases, G protein-coupled receptors (GPCRs), and the Ras superfamily of small G proteins. A major challenge is to decipher how each PI3K isoform is able to successfully synergize these inputs into their intended signaling function. This article highlights recent progress in characterizing the molecular mechanisms of PI3K isoform-specific activation pathways, as well as novel roles for PI3Ks in human diseases, specifically cancer and immune diseases.

Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α

Summary Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.