Shane Minogue

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor

The type II alpha isoform of phosphatidylinositol 4-kinase has recently been shown to function in the recruitment of adaptor protein-1 complexes to the trans-Golgi network. Here we show that phosphatidylinositol 4-kinase IIα is also a component of highly dynamic membranes of the endosomal system where it colocalises with protein markers of the late endosome and with endocytosed epidermal growth factor. When phosphatidylinositol 4-kinase IIα activity was inhibited in vivo using the monoclonal antibody 4C5G or by depression of endogenous phosphatidylinositol 4-kinase IIα protein levels using RNA interference, ligand-bound epidermal growth factor receptor failed to traffic to late endosomes and instead accumulated in vesicles in a sub-plasma membrane compartment. Furthermore, lysosomal degradation of activated epidermal growth factor receptor was dramatically impaired in small inhibitory RNA-treated cells. We demonstrate that phosphatidylinositol 4-kinase IIα is necessary for the correct endocytic traffic and downregulation of activated epidermal growth factor receptor.

Oxysterol Binding Protein-dependent Activation of Sphingomyelin Synthesis in the Golgi Apparatus Requires Phosphatidylinositol 4-Kinase IIα

The study identifies a sterol- and oxysterol binding protein (OSBP)-regulated phosphatidylinositol 4-kinase that regulates ceramide transport protein (CERT) activity and sphingomyelin (SM) synthesis. RNA interference silencing experiments identify PI4KIIα; as the mediator of Golgi recruitment of CERT, providing a potential mechanism for coordinating assembly of SM and cholesterol in the Golgi or more distal compartments.

Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum

Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.

Loss of phosphatidylinositol 4-kinase 2α activity causes late onset degeneration of spinal cord axons

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2α isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2α kinase activity initially appear normal. However, adult Pi4k2aGT/GT animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2aGT/GT animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

Type II phosphatidylinositol 4-kinase IIα (PI4KIIα) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIα has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIα mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIα activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIα to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIα demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIα was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIα. Further measurements revealed that the reduction in the mobile fraction of PI4KIIα correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.

Lipid and Peptide Control of Phosphatidylinositol 4-Kinase IIα Activity on Golgi-endosomal Rafts

The most abundant and widely expressed mammalian phosphoinositide kinase activity is contributed by phosphatidylinositol 4-kinase IIα (PI4KIIα). In this study we demonstrate that PI4KIIα is a novel GTP-independent target of the wasp venom tetradecapeptide mastoparan and that different mechanisms of activation occur in different subcellular membranes. Following cell membrane fractionation mastoparan specifically stimulated a high activity Golgi/endosomal pool of PI4KIIα independently of exogenous guanine nucleotides. Conversely, GTPγS stimulated a low activity pool of PI4KIIα in a separable dense membrane fraction and this response was further enhanced by mastoparan. Overexpression of PI4KIIα increased the basal phosphatidylinositol 4-kinase activity of each membrane pool, as well as the mastoparan-dependent activities, thereby demonstrating that mastoparan specifically activates this isozyme. Both mastoparan and M7, at concentrations known to invoke secretion, stimulated PI4KIIα with similar efficacies, resulting in an increase in the apparent Vmax and decrease in Km for exogenously added PI. Mastoparan also stimulated PI4KIIα immunoprecipitated from the raft fraction, indicating that PI4KIIα is a direct target of mastoparan. Finally we reveal a striking dependence of both basal and mastoparan-stimulated PI4KIIα activity on endogenous cholesterol concentration and therefore conclude that changes in membrane environment can regulate PI4KIIα activity.

Pathogenetic mechanisms of amyloid A amyloidosis

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.

Mammalian phosphatidylinositol 4-kinases as modulators of membrane trafficking and lipid signaling networks.

Abstract The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and such structural differences determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during distinct stages of GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.