66 research outputs found
Characterization of Class D VPS Proteins
No full textThe vacuole of the yeast Saccharomyces cerevisiae is functionally similar to the mammalian lysosome. The components of the VPS (vacuolar protein sorting) system are responsible for proper delivery of vacuolar biosythetic enzymes. Efforts to dissect the genetics of this system have revealed several classes of mutants, each defective in one transport step in the VPS pathway. The Class D VPS proteins are thought to control anterograde traffic between the late Golgi and late endosome. Although most of these proteins have homologues of known function in other systems, two exceptions are the Vps3p and Vps8p proteins. Analysis of Vps3p reveals that it is associated with a highdensity structure, possibly a coated vesicle or a large protein complex. The Vps8p protein contains a C-terminal H2 RING finger motif, a domain often associated with E3 ubiquitin ligase activity. In vitro analysis reveals that a Vps8p fragment containing this domain has this activity. Deletion of the RING finger reveals that the endocytic marker Ste3p accumulates in an abnormally large late-endosome-derived structure, but that sorting of the soluble vacuolar cargo CPY is relatively unaffected. These results suggest a division of function within the Vps8p molecule
Role of Ribavirin, an Anti-Viral Agent, In Eukaryotic Initiation Factor 4e (Eif4e) Subcellular Localization and Function
No full textExpression of eukaryotic initiation factor 4E (eIF4E) is necessary for active cellular growth and catalysis of the rate-limiting step in cap-dependent protein synthesis3-5. Studies in the past decade have shown that eIF4E contributes to malignancy by selectively facilitating the translation of a specific set of mRNAs, those that generally encode key proteins involved in cellular growth, angiogenesis and survival6-9. Key questions remaining in the field are: (1) what is the mechanism by which eIF4E levels are increased in tumor cells? (2) What is the role of eIF4E in regulating the translation of gene products involved in various aspects of malignancy? Finally, (3) how can eIF4E be exploited as a therapeutic target for human cancer progression? Recently, Kentsis and colleagues10 demonstrated that ribavirin disrupts subcellular organization of eIF4E and suppresses eIF4E-mediated oncogenic transformation of murine cells and tumor growth of a mouse model of eIF4E-dependent human squamous cell carcinoma. These findings suggest a specific and novel mechanism by which ribavirin affects cellular distribution and function of eIF4E, a topic that will be explored in the current proposal. This investigation may facilitate understanding in the regulation of transcription and thus provide a potential strategy for interrupting oncogenic cascades
Distinct Functional Phases in Proteins: A Test by Large-Scale Protein Design
No full textNote: The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access only.The biological properties of proteins - folding, biochemical functions, and evolvability - originate from the global pattern of interactions between amino acids. Coevolution studies suggest a model for this pattern in which the essential constraints are loaded in sparse networks of cooperative residues (termed sectors), embedded within an environment of weakly coupled residues. Here, we test this biphasic model for proteins using a protein design approach in the SHO1-mediated yeast osmo-sensing pathway. We computationally designed libraries of synthetic SHO1 SH3 domains in which the hierarchy of coevolution that defines sectors and their environment is gradually varied. We tested the designed sequences in a quantitative high-throughput assay for SHO1 function in vivo. The data show that sector amino acids contribute in an all-or-nothing fashion while surrounding amino acids have a more graded, near-independent contribution to function. These results support the biphasic model for the information content of protein sequences
Spindle Checkpoint at Kinetochores
No full textThe kinetochore—a large protein assembly on centromeric chromatin—functions as the docking site for spindle microtubules and as a signaling hub for the spindle checkpoint. The Constitutive Centromere-Associated Network (CCAN) at the inner kinetochore nucleates the formation of the mature outer kinetochore during mitosis, including the recruitment of the KMN network that consists of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The KMN is a critical receptor for microtubules, and provides a landing pad for various spindle checkpoint proteins and regulatory factors. The spindle checkpoint protein Mad2 has multiple conformations, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). The kinetochore-bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of O-Mad2 and serves as a catalytic engine of checkpoint signaling. The activated C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Deficient spindle checkpoint signaling leads to premature sister-chromatid separation and aneuploidy. Research in this thesis has provided several key insights into spindle checkpoint signaling at kinetochores.
First, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein do not form C-Mad2 on their own. Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20. Our results establish for the first time that the conformational change of Mad2 is regulated by posttranslational mechanisms.
Second, we have studied how Mad1 is targeted to kinetochores. We have determined the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. The structure reveals unexpected fold similarity between Mad1 CTD and known kinetochore-binding modules. Functional studies then validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Interestingly, deletion of the CTD does not abolish Mad1 kinetochore localization. Non-overlapping Mad1 fragments retain detectable kinetochore targeting. Our results indicate that the CTD–Bub1 connection is one of several mechanisms of targeting Mad1 to kinetochores.
Finally, we show that the proper assembly of KMN is required for generating the spindle checkpoint signal at kinetochores. We have developed several strategies to inactivate KMN at kinetochores in human cells, and demonstrate its requirement for the spindle checkpoint in the absence of microtubules. We further show that two quasi-independent pathways mediate the mitosis-specific assembly of KMN at kinetochores. In one pathway, the centromeric kinase Aurora B phosphorylates the Mis12C component Dsn1, and strengthens Mis12C binding to the CCAN component CENP-C. In the second pathway, CENP-T anchors the CENP-H/I/K sub-complex at kinetochores, which in turn recruits Ndc80C. Inactivation of both pathways abolishes KMN at kinetochores and causes gross spindle checkpoint defects.
In conclusion, combining cell biology and structural biology methods, our studies have defined a new posttranslational mechanism of Mad2 regulation, uncovered a critical way for targeting Mad1 to kinetochores, and dissected assembly pathways of the KMN checkpoint sensor at kinetochores
Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress
No full textThe post-translational modification AMPylation is emerging as a significant regulatory mechanism in both prokaryotic and eukaryotic biology. This process involves the covalent addition of an adenosine monophosphate to a protein resulting in a modified protein with altered activity. Proteins capable of catalyzing AMPylation, termed AMPylators, are comparable to kinases in that they both hydrolyze ATP and reversibly transfer a part of this primary metabolite to a hydroxyl side chain of the protein substrate. To date, all AMPylators discovered contain one of two domains: the Fic domain or the adenylyl transferase domain.
All currently characterized AMPylators are bacterial in origin and are primarily Type III or Type IV secreted effector proteins, which are injected into a host cell to manipulate host signaling to the microbe's advantage. Examples of these are VopS (Vibrio parahaemolyticus), IbpA (Histophilus somni) and DrrA (Legionella pneumophila). The discovery of SidD, a deAMPylator also from L. pneumophila, shows that this modification is dynamic and could likely have a regulatory role in eukaryotic biology. Supporting this idea is the presence of a single copy of the Fic domain in most metazoans, including humans. The substrates, localization, and function of Fic proteins and other AMPylators in eukaryotic biology are perhaps the largest open questions in this rapidly expanding field.
The goal of my dissertation work was to expand the understanding of the effects of AMPylation in eukaryotic signaling. I approached this goal in three ways: by examining the effects of an AMPylator (VopS) with known targets (Rho GTPases) on different aspects of cell signaling, developing screening tools for AMPylation and attempting to elucidate some of the functions of the human AMPylator, FicD, in which the targets are unclear. I found that VopS, in addition to collapsing the host actin cytoskeleton, also inhibits many aspects of host defense signaling including NFB, MAP kinases and the phagocytic NADPH oxidase system. I explored the possibility of other potential substrates of VopS by collaborating on an extensive protein microarray screen for AMPylation, determining that the entire Rho GTPase family is AMPylated. I also discovered that the human AMPylator FicD is induced during the unfolded protein response, is localized to the endoplasmic reticulum and is capable of AMPylating the ER chaperone BiP/GRP78. The progress made in these studies will contribute to understanding the role of this enigmatic modification in mammalian cell signaling
Regulation of p190RhoGEF by Activated Rho and Rac GTPases: Amplification and Crosstalk
No full textThe Rho family of monomeric GTPases regulates a wide range of cellular processes including cytoskeletal structure, motility, cell division, gene transcription, vesicular transport, and various enzymatic activities. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs), which catalyze the exchange of GTP for GDP on Rho. For classical RhoGEFs, the exchange activity resides in a Dbl homology (DH) domain, which is linked to a pleckstrin homology (PH) domain that subserves various functions. We have crystallized and solved structures of the PH domain from p190RhoGEF bound to either RhoA•GTP or Rac1•GTP. The interfaces between activated Rac1 or RhoA with the PH domain utilize the same hydrophobic surface on the PH domain. Similar to RhoA, activated Rac1 interacts with the PH domain via its effector-binding surface albeit burying less surface area than the interaction of RhoA with the PH domain. Consequently, the PH domain of p190RhoGEF has a higher affinity for RhoA than Rac1. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of the p190RhoGEF to the substrate, RhoA•GDP, in vitro; mutations of key hydrophobic residues in the PH domain abolish this stimulation. Among members of the homologous Lbc subfamily of RhoGEFs, p190RhoGEF exhibited the greatest capacity for regulation by Rac1. While interaction of RhoA with the PH domain provides a mechanism for direct positive feedback, the novel interaction of activated Rac1 with the PH domain of p190RhoGEF reveals a potential mechanism for cross-talk regulation between the Rho and the Rac signaling pathways
Dissecting the Role of the Lipodystrophy Protein Seipin in the Biogenesis of the Lipid Droplet Organelle
No full textLong thought to be little more than inert storage depots, lipid droplets have recently become recognized as unique, dynamic, regulated organelles that play an essential role in fat storage. Despite this increased interest, much remains unknown. Lipid droplets have been observed to emerge from the endoplasmic reticulum, but the available models for lipid droplet biogenesis are largely conceptual, with little to no evidence for specific mechanisms of droplet formation. Debate even continues within the field as to whether lipid droplet formation is a spontaneous process, driven by physicochemical and hydrophobic forces, or a regulated process driven by protein factors.
The Goodman laboratory previously found evidence to suggest that seipin, mutated in the most severe cases of congenital generalized lipodystrophy, may be a key factor in the early stages of lipid droplet formation. Seipin resides at the junction between lipid droplets and the endoplasmic reticulum, and deletion of seipin results in both a drastic impediment to de novo droplet formation and a striking disorganization of droplet morphology.
For my thesis work, I have explored several aspects of seipin’s role at the lipid droplet. I have studied the effects of seipin deletion on protein targeting to abnormal lipid droplets, through which I identified a unique effect of seipin on the regulation of lipase targeting. I have also analyzed the topology of the seipin complex itself through a series of deletion mutants, identifying regions that contribute to the localization, membrane association, and stability of the seipin complex. Furthermore, these studies have led to novel insights on the function of seipin, through the characterization of a remarkable N-terminal seipin mutation that presents with defects in droplet initiation but homogenous droplet morphology. I have therefore concluded that seipin plays two dissectible roles in lipid droplet formation: 1) promoting lipid droplet initiation and 2) regulating subsequent droplet morphology. Finally, I suggest hypotheses on the mechanisms by which seipin exerts these effects, proposing that the N-terminus of seipin may regulate lipin, a mouse lipodystrophy protein, to effect droplet initiation, while the bulk of the protein may serve to regulate the access of phospholipids to the lipid droplet surface
The Assembly of Pathogenic Signaling Circuits by a Family of Bacterial Secreted Effector Proteins
No full textBacterial type III secreted effector proteins facilitate Gram-negative bacterial replication, dissemination, and immune evasion in the infected host organism. While much attention has been focused on the cell inhibitory mechanisms of these virulence factors, there is emerging evidence that bacterial effectors exert direct control over host cellular behavior by assembling new signaling circuits from pre-existing regulatory modules. However, these mechanisms are poorly understood. In this work, we utilize the WxxxE family of effector proteins as a model system to understand how pathogens rewire host signaling cascades. These effectors share a core catalytic domain that functions as a guanine-nucleotide exchange factor (GEF) for Rho family GTPases. Using a structure to function approach, we uncover a GEF-GTPase pairing mechanism important for signaling fidelity and pathogenic diversity. Guided by these structural insights, we next wanted to know how E. coli, an extracellular pathogen, induces the polarization of host actin molecules. By using synthetic derivatives of the enteropathogenic E. coli GEF Map, we discover that Cdc42 GTPase activity cycles are controlled in space and time by Map’s interaction with F-actin. Mathematical modeling reveals how actin dynamics coupled to a Map-dependent positive feedback loop spontaneously polarizes Cdc42. By reconstituting the system, we further show how cells polarize in response to an extracellular spatial cue. These results demonstrate how pathogens gain systems level control over host signaling networks and suggest a new view of cellular polarity centered on the interaction between GEFs and F-actin. To explore alternative mechanisms that bacteria utilize to assemble circuits, we utilize yeast genetics to identify novel membrane-interactions. We identify for the first time the direct association of the Shigella GEF IpgB1 with acidic phospholipids. Surprisingly, we find that these protein-lipid interactions are not required for IpgB1’s known role in Shigella invasion. However, we do find that IpgB1’s interactions with eukaryotic membranes are essential for bacterial replication and persistence within host cells. Furthermore, we identify a pathogenic circuit that connects GTPase activity with phospholipid metabolism. In summation, our findings illustrate the complex evolutionary relationship between pathogen and host, and how investigating these interactions provide insight into endogenous signaling systems
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