Biochemistry and Pharmacology - Research Publications
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ItemThe trans-Golgi network is a major site for α-secretase processing of amyloid precursor protein in primary neurons(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-02-01)Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase-mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor-like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.
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ItemThe Function of the Golgi Ribbon Structure - An Enduring Mystery Unfolds!(WILEY, 2017-11)The Golgi apparatus in vertebrate cells consists of individual Golgi stacks fused together in a continuous ribbon structure. The ribbon structure per se is not required to mediate the classical functions of this organelle and the relevance of the "ribbon" structure has been a mystery since first identified ultrastructurally in the 1950s. Recent advances recognize a role for the Golgi apparatus in a range of cellular processes, some mediated by signaling networks which are regulated at the Golgi. Here we review the cellular processes and signaling events regulated by the Golgi apparatus and, in particular, explore an emerging theme that the ribbon structure of the Golgi contributes directly to the regulation of these higher order functions.
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ItemAmyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b-and AP4-dependent pathway(WILEY, 2017-03)The intracellular trafficking and proteolytic processing of the membrane-bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid-beta (Aβ) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by β-secretase (BACE1) and γ-secretase. Here, we show that the majority of newly synthesized APP is transported from the trans-Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post-Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aβ secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aβ.
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ItemIncreased endogenous antigen presentation in the periphery enhances susceptibility to inflammation-induced gastric autoimmunity in mice(WILEY-BLACKWELL, 2017-01)How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self-reactive T cells (A23 T cells) specific for the gastric H+ /K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild-type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE-H+ /K+ β transgenic mice (IEβ) reduces A23 T-cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα-specific A23 T cells from immunised wild-type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEβ transgenic mice were readily re-activated, indicating loss of T-cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self-reactive T cells is critical in the balance between tolerance and autoimmunity.
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ItemForm and function of the Golgi apparatus: scaffolds, cytoskeleton and signalling(WILEY, 2019-09)In addition to the classical functions of the Golgi in membrane transport and glycosylation, the Golgi apparatus of mammalian cells is now recognised to contribute to the regulation of a range of cellular processes, including mitosis, DNA repair, stress responses, autophagy, apoptosis and inflammation. These processes are often mediated, either directly or indirectly, by membrane scaffold molecules, such as golgins and GRASPs which are located on Golgi membranes. In many cases, these scaffold molecules also link the actin and microtubule cytoskeleton and influence Golgi morphology. An emerging theme is a strong relationship between the morphology of the Golgi and regulation of a variety of signalling pathways. Here, we review the molecular regulation of the morphology of the Golgi, especially the role of the golgins and other scaffolds in the interaction with the microtubule and actin networks. In addition, we discuss the impact of the modulation of the Golgi ribbon in various diseases, such as neurodegeneration and cancer, to the pathology of disease.
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ItemGGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production(AMER SOC CELL BIOLOGY, 2018-01-15)The diversion of the membrane-bound β-site amyloid precursor protein-(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production.
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ItemExpression of HIV-1 Vpu Leads to Loss of the Viral Restriction Factor CD317/Tetherin from Lipid Rafts and Its Enhanced Lysosomal Degradation(PUBLIC LIBRARY SCIENCE, 2013-09-24)CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.
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ItemRab6a/a′ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages(PUBLIC LIBRARY SCIENCE, 2013-02-21)Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.
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ItemSNX5 is essential for efficient macropinocytosis and antigen processing in primary macrophages(COMPANY OF BIOLOGISTS LTD, 2012-09-15)Macropinocytosis mediates the bulk endocytosis of solute molecules, nutrients and antigens. As this endocytic pathway is considered important in functions associated with immune responses, the molecular mechanisms regulating this pathway in immune cells is of particular significance. However, the regulators of macropinocytosis in primary cells remain poorly defined. Members of the sorting nexin (SNX) family have been implicated in macropinosome biogenesis in cultured cells and here we have analyzed the role of two SNX family members, SNX1 and its binding partner SNX5, in macropinocytosis of mouse primary macrophages. We show that endogenous SNX1 and SNX5 are localised to newly-formed macropinosomes in primary mouse macrophages and, moreover, demonstrate that SNX5 plays an essential role in macropinosome biogenesis. Depletion of SNX5 in bone marrow-derived macrophages dramatically decreased both the number and size of macropinosomes. Depletion of SNX5 also resulted in dramatic reduction in uptake and processing of soluble ovalbumin in macrophages, indicating that the majority of antigen uptake and delivery to late endosomes is via macropinocytosis. By contrast, the absence of SNX1 had no effect on endogenous SNX5 localisation and macropinosome biogenesis using macrophages from SNX1 knockout mice. Therefore, SNX5 can function independently of SNX1 and is a modulator of macropinocytosis that influences the uptake and processing of soluble antigen in primary mouse macrophages.