Stacking Protein Research Articles

Abstract 507: Nitrogen oxides-derived immunotherapy: Revealing cryptic melanoma-specific immune targets

Abstract Nitrogen oxides (NOx) can exert their relevant biological functions through two reversible molecular modifications: a) direct interaction with metals (nitrosation), and b) by interacting with thiols (s-nitrosylation); or irreversible modifications of aromatic amino acid residues such as tyrosine or phenylalanine (nitration). Any of these modifications have been demonstrated to affect the biological fate of the modified molecule or alter their functions. We have previously shown that these NOx-derived changes will trigger a myriad of modifications at the genetic, epigenetic and post-translational level in tumor cells resulting in a robust and effective therapeutic immune response against cancer cells. However, the molecular mechanisms and immune-related tumor targets remain elusive. Thus, we hypothesize that NOx-derived protein modifications (i.e., Tyrosine nitration) will differentially prime the immune system with otherwise subdominant antigens (cryptic) making them immune visible and triggering an effective anti-tumor immune response. We have tested this hypothesis using the well-characterized B16-C57BL/6 syngeneic and poorly immunogenic melanoma mouse model. NOx-modified and non-modified B16-F0 melanoma cell lysates were used to immunize naïve C57BL/6. Post-immunized sera was screened against SDS-PAGE resolved untreated B16-F0 lysates and human protein library micro array for differential recognition of immune cross-reactive of novel mouse and human melanoma targets by the mouse IgG fraction. Our results demonstrated the highly significant (p <0.0001) IgG-associated immune recognition of five cross-reactive novel melanoma targets (in relative affinity decreasing order): 1) Golgi reassembly stacking protein 1 (GORASP1), 2) ArfGAP w/GTPase domain-ankyrin repeat and PH domain 1 (AGAP1), 3) Microtubule-associated protein tau (MAPT), 4) Mitochondrial ribosomal protein L46 (MRPL46), and 5) Protocadherin beta 6 (PCDHB6). Currently, we are evaluating the significance and functional activity of the identified targets and their isolated/purified specific immune effector (IgG) counterparts. In addition, we are characterizing the potential molecular and immunological mechanisms that might lead to the use of these novel melanoma-specific immune targets as diagnostic/prognostic and/or immunotherapeutic tools. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 507. doi:1538-7445.AM2012-507

Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β.

Autophagy controls the quality and quantity of the eukaryotic cytoplasm while performing two evolutionarily highly conserved functions: cell-autonomous provision of energy and nutrients by cytosol autodigestion during starvation, and removal of defunct organelles and large aggregates exceeding the capacity of other cellular degradative systems. In contrast to these autodigestive processes, autophagy in yeast has additional, biogenesis functions. However, no equivalent biosynthetic roles have been described for autophagy in mammals. Here, we show that in mammalian cells, autophagy has a hitherto unappreciated positive contribution to the biogenesis and secretion of the proinflammatory cytokine IL-1β via an export pathway that depends on Atg5, inflammasome, at least one of the two mammalian Golgi reassembly stacking protein (GRASP) paralogues, GRASP55 (GORASP2) and Rab8a. This process, which is a type of unconventional secretion, expands the functional manifestations of autophagy beyond autodigestive and quality control roles in mammals. It enables a subset of cytosolic proteins devoid of signal peptide sequences, and thus unable to access the conventional pathway through the ER, to enter an autophagy-based secretory pathway facilitating their exit from the cytoplasm.

Adaptor Protein-3 (AP-3) Complex Mediates the Biogenesis of Acidocalcisomes and Is Essential for Growth and Virulence of Trypanosoma brucei

Acidocalcisomes are acidic calcium and polyphosphate storage organelles found in a diverse range of organisms. Here we present evidence that the biogenesis of acidocalcisomes in Trypanosoma brucei is linked to the expression of adaptor protein-3 (AP-3) complex. Localization studies in cell lines expressing β3 and δ subunits of AP-3 fused to epitope tags revealed their partial co-localization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes, with the Golgi marker Golgi reassembly and stacking protein, and with antibodies against the small GTPase Rab11. Ablation of the β3 subunit by RNA interference (RNAi) resulted in disappearance of acidocalcisomes from both procyclic and bloodstream form trypanosomes, as revealed by immmunofluorescence and electron microscopy assays, with no alterations in trafficking of different markers to lysosomes. Knockdown of the β3 subunit resulted in lower acidic calcium, pyrophosphate, and polyphosphate content as well as defects in growth in culture, resistance to osmotic stress, and virulence in mice. Similar results were obtained by knocking down the expression of the δ subunit of AP-3. These results indicate that AP-3 is essential for the biogenesis of acidocalcisomes and for growth and virulence of T. brucei.

Unravelling Secretion in Cryptococcus neoformans: More than One Way to Skin a Cat

Secretion pathways in fungi are essential for the maintenance of cell wall architecture and for the export of a number of virulence factors. In the fungal pathogen, Cryptococcus neoformans, much evidence supports the existence of more than one route taken by secreted molecules to reach the cell periphery and extracellular space, and a significant degree of crosstalk between conventional and non-conventional secretion routes. The need for such complexity may be due to differences in the nature of the exported cargo, the spatial and temporal requirements for constitutive and non-constitutive protein secretion, and/or as a means of compensating for the extra burden on the secretion machinery imposed by the elaboration of the polysaccharide capsule. This review focuses on the role of specific components of the C. neoformans secretion machinery in protein and/or polysaccharide export, including Sec4, Sec6, Sec14, Golgi reassembly and stacking protein and extracellular exosome-like vesicles. We also address what is known about traffic of the lipid, glucosylceramide, a target of therapeutic antibodies and an important regulator of C. neoformans pathogenicity, and the role of signalling pathways in the regulation of secretion.

Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence

Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.

The multiple facets of the Golgi reassembly stacking proteins

The multiple facets of the Golgi reassembly stacking proteins

The multiple facets of the Golgi reassembly stacking proteins

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-XL as a candidate apoptotic binding partner for GRASP65.

Golgi reassembly stacking protein 55 interacts with membrane‐type (MT) 1‐matrix metalloprotease (MMP) and furin and plays a role in the activation of the MT1‐MMP zymogen

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a proteinase involved in the remodelling of extracellular matrix and the cleavage of a number of substrates. MT1-MMP is synthesized as a zymogen that requires intracellular post-translational cleavage to gain biological activity. Furin, a member of the pro-protein convertase family, has been implicated in the proteolytic removal of the MT1-MMP prodomain sequence. In the present study, we demonstrate a role for the peripheral Golgi matrix protein GRASP55 in the furin-dependent activation of MT1-MMP. MT1-MMP and furin were found to co-localize with Golgi reassembly stacking protein 55 (GRASP55). Further analysis revealed that GRASP55 associated with the cytoplasmic domain of both proteases and that the LLY(573) motif in the MT1-MMP intracellular domain was crucial for the interaction with GRASP55. Overexpression of GRASP55 was found to enhance the formation of a complex between MT1-MMP and furin. Finally, we report that disruption of the interaction between GRASP55 and furin led to a reduction in pro-MT1-MMP activation. Taken together, these data suggest that GRASP55 may function as an adaptor protein coupling MT1-MMP with furin, thus leading to the activation of the zymogen.

The Yeast GRASP Grh1 Colocalizes with COPII and Is Dispensable for Organizing the Secretory Pathway

In mammalian cells, the 'Golgi reassembly and stacking protein' (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non-essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER-Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER-Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)-binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2-like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.

Ultrastructural Study of Golgi Duplication in Trypanosoma brucei

Golgi duplication in the protozoan parasite Trypanosoma brucei has been tracked using serial thin section three-dimensional reconstructions of transmission electron micrographs. The old Golgi maintains a constant size (approximately 0.060 microm(3)) throughout the cell cycle. A morphologically identifiable new Golgi appears at approximately 0.20 of the cell cycle (defined by the size of the nucleus and lasting about 9 h) and grows from approximately 0.018 microm(3) until it is the same size as the old Golgi (by approximately 0.55 of the cell cycle). Morphologically identifiable late Golgi appear at approximately 0.58 of the cell cycle, but their volume ( approximately 0.036 microm(3)) did not change significantly. Cryoimmunoelectron microscopy was used to identify candidates for the earliest new Golgi structures, and these comprised clusters of vesicles containing Golgi reassembly stacking protein (GRASP) near an endoplasmic reticulum exit site. These results, combined with earlier fluorescence data, suggest that the new Golgi begins functioning before cisternal stacks are formed.

Pentameric Assembly of Potassium Channel Tetramerization Domain-Containing Protein 5

We report the X-ray crystal structure of human potassium channel tetramerization domain-containing protein 5 (KCTD5), the first member of the family to be so characterized. Four findings were unexpected. First, the structure reveals assemblies of five subunits while tetramers were anticipated; pentameric stoichiometry is observed also in solution by scanning transmission electron microscopy mass analysis and analytical ultracentrifugation. Second, the same BTB ( bric-a-brac, tramtrack, broad complex) domain surface mediates the assembly of five KCTD5 and four voltage-gated K + (Kv) channel subunits; four amino acid differences appear crucial. Third, KCTD5 complexes have well-defined N- and C-terminal modules separated by a flexible linker that swivels by ∼ 30 o; the C-module shows a new fold and is required to bind Golgi reassembly stacking protein 55 with ∼ 1 μM affinity, as judged by surface plasmon resonance and ultracentrifugation. Fourth, despite the homology reflected in its name, KCTD5 does not impact the operation of Kv4.2, Kv3.4, Kv2.1, or Kv1.2 channels.

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.

Molecular Mechanism of Mitotic Golgi Disassembly and Reassembly Revealed by a Defined Reconstitution Assay

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.

Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

Spatial dissection of the cis‐ and trans‐Golgi compartments in the malaria parasite Plasmodium falciparum

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis-side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of PfSec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis- to trans-Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans-Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.

The Golgi-Associated Protein GRASP Is Required for Unconventional Protein Secretion during Development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.