Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.

Phox homology (PX) domains are named for a 130-amino acid region of homology shared with part of two components of the phagocyte NADPH oxidase (phox) complex. They are found in proteins involved in vesicular trafficking, protein sorting, and lipid modification. It was recently reported that certain PX domains specifically recognize phosphatidylinositol 3-phosphate (PtdIns-3-P) and drive recruitment of their host proteins to the cytoplasmic leaflet of endosomal and/or vacuolar membranes where this phosphoinositide is enriched. We have analyzed phosphoinositide binding by all 15 PX domains encoded by the Saccharomyces cerevisiae genome. All yeast PX domains specifically recognize PtdIns-3-P in protein-lipid overlay experiments, with just one exception (a significant sequence outlier). In surface plasmon resonance studies, four of the yeast PX domains bind PtdIns-3-P with high (micromolar range) affinity. Although the remaining PX domains specifically recognize PtdIns-3-P, they bind this lipid with only low affinity. Interestingly, many proteins with "low affinity" PX domains are known to form large multimeric complexes, which may increase the overall avidity for membranes. Our results establish that PtdIns-3-P, and not other phosphoinositides, is the target of all PX domains in S. cerevisiae and suggest a role for PX domains in assembly of multiprotein complexes at specific membrane surfaces.The phox homology (PX) 1 domain (1) was first identified in 1996 as a 130-amino acid region of homology present in two components (p40 phox and p47 phox ) of the phagocyte NADPH oxidase (phox) complex plus a wide variety of other proteins with diverse functions. Many PX domain-containing proteins are involved in vesicular trafficking, protein sorting, and lipid modification. The presence of a central PXXP motif in most (but not all) PX domains stimulated the initial suggestion that they represent binding partners for SH3 domains (1) and therefore may direct inter-and/or intramolecular protein-protein interactions. In support of this, the PX domain from p47 phox was recently shown to interact, albeit weakly, with the C-terminal SH3 domain from the same protein (2).A different class of PX domain ligand was recently reported by several groups who described binding of PX domains to phosphoinositides. Specifically, the PX domains from the yeast vacuolar t-SNARE Vam7p (3, 4), sorting nexin-3 (SNX3) (5), and p40 phox (6, 7) were all shown to bind selectively to phosphatidylinositol 3-phosphate (PtdIns-3-P), which is enriched in endosomal and vacuolar membranes (8). Isolated PX domains from p40 phox and SNX3 were found to be independently capable of localizing to endosomal structures in vivo in a manner that depends upon their ability to bind PtdIns-3-P and on PI-3-kinase activity (5-7). Similarly, analysis of deletion mutants indicated that the Vam7p PX domain is sufficient for localization of that protein to vacuoles and endosomes in yeast (3). Other phosphoinositides have been reported as the preferred ligands for certain PX domain...

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Cellular FLICE-inhibitory protein (c-FLIPL) is a key regulator of the extrinsic cell death pathway. Although widely regarded as an inhibitor of initiator caspase activation and cell death, c-FLIPL is also capable of enhancing procaspase-8 activation through heterodimerization of their respective protease domains. However, the underlying mechanism of this activation process remains enigmatic. Here, we demonstrate that cleavage of the intersubunit linker of c-FLIPL by procaspase-8 potentiates the activation process by enhancing heterodimerization between the two proteins and vastly improving the proteolytic activity of unprocessed caspase-(C)8. The crystal structures of the protease-like domain of c-FLIPL alone and in complex with zymogen C8 identify the unique determinants that favor heterodimerization over procaspase-8 homodimerization, and induce the latent active site of zymogen C8 into a productive conformation. Together, these findings provide molecular insights into a key aspect of c-FLIPL function that modulates procaspase-8 activation to elicit diverse responses in different cellular contexts.apoptosis ͉ caspase-8 ͉ extrinsic pathway ͉ cellular FLICE-inhibitory protein A poptosis is a cellular suicide program essential for early development, tissue homeostasis, and immune system maintenance in all metazoans (1, 2). This program is executed by a family of cysteine proteases known as caspases, which are synthesized as latent zymogens, and are activated in a hierarchical manner as initiators and effectors of the cell death process (3). In the extrinsic cell death pathway, ligands such as FasL/CD95L directly engage their cognate death receptors at the cell surface to recruit and activate the initiator caspases (4). This process occurs through homotypic death domain (DD) interactions between the intracellular portion of the Fas receptor and the adaptor protein FADD, and homotypic death effector domain (DED) interactions between FADD and the N terminus of procaspase-8 or procaspase-10. The resulting activation platform constitutes the death-inducing signaling complex (DISC) (5), effectively homo-oligomerizing and activating the protease domains of procaspase-8 and procaspase-10. Once activated and processed to the mature form, these initiator caspases then cleave and activate the effector caspases, which ultimately proteolyze and dismantle the contents of the cell.During the early stages of death receptor signaling, the key regulatory proteins c-FLIP L , c-FLIP S , and c-FLIP R are also recruited to the DISC, where they modulate activation of procaspase-8 and procaspase-10 (6, 7). The long form of FLIP (c-FLIP L ) is highly homologous to procaspase-8 and procaspase-10, possessing the same architecture with tandem DEDs at its N terminus (the prodomain) and a protease-like domain at its C terminus that is catalytically inactive. The short forms (c-FLIP S and c-FLIP R ) by contrast primarily contain the N-terminal DEDs. All forms of FLIP are widely accepted as antiapoptotic proteins that compete with procaspase-8 ...

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

The mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, a key component of the Carma1/Bcl10/ MALT1 signalosome, is critical for NF-κB signaling in multiple contexts. MALT1 is thought to function as a scaffold and protease to promote signaling; however, the biochemical and structural basis of paracaspase action remains largely unknown. Here we report the 1.75-Å resolution crystal structure of the MALT1 paracaspase region, which contains the paracaspase domain and an ensuing Ig-like domain. The paracaspase and the Ig domains appear as a single folding unit and interact with each other through extensive van der Waals contacts and hydrogen bonds. The paracaspase domain adopts a fold that is nearly identical to that of classic caspases and homodimerizes similarly to form an active protease. Unlike caspases, the active and mature form of the paracaspase domain remains a single uncleaved polypeptide and specifically recognizes the bound peptide inhibitor Val-Arg-Pro-Arg. In particular, the carboxyl-terminal amino acid Arg of the inhibitor is coordinated by three highly conserved acidic residues. This structure serves as an important framework for deciphering the function and mechanism of paracaspases exemplified by MALT1.T he transcription factor NF-κB is a key constituent of all cell types and is activated by various receptors to regulate survival, proliferation, migration, and differentiation (1). In particular, NF-κB functions early in the development and maintenance of innate and adaptive immune systems and execution of the immune response. Although caspases, cysteine proteases that cleave substrate proteins after aspartate residues, are widely known as executioners of programmed cell death or apoptosis (2), a subset and related members also activate NF-κB to promote lymphocyte proliferation and inflammation. One such caspase-like family member, the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, was identified through weak sequence homology to caspases (3) and was subsequently found to play an important role in lymphocyte activation (4) and disease progression in MALT lymphomas (5).Upon antigen receptor stimulation, the MALT1 paracaspase and Bcl10 assemble into the Carma1/Bcl10/MALT1 (CBM) signalosome to activate NF-κB in the adaptive immune system. Specifically during T-cell receptor signaling, the CBM signalosome is thought to oligomerize MALT1 and its associated ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) or TRAF2 (6, 7), which in turn facilitates K63-linked polyubiquitylation of multiple proteins including the regulatory γ-subunit of the IκB kinase (IKK) complex (6, 7), TRAF6 itself (7), Bcl10 (8), and MALT1 (9). Poly ubiquitylation of these proteins ultimately leads to the recruitment of transforming growth factor β-activated kinase 1 (TAK1), TAK1 binding protein (TAB), and the IKK complex to lipid rafts where the IKKβ-subunit is phosphorylated and activated. In the canonical NF-κB pathway, the activated IKK complex ph...