Browsing by Subject "Microtubule-Associated Proteins"
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Item Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item Insight into Microtubule Catastrophe and Growth(2020-08-01T05:00:00.000Z) Kim, Tae Hyungmin; Lin, Milo; Jaqaman, Khuloud; Rice, Luke M.; Yu, HongtaoMicrotubules are hollow cylindrical polymers of αβ-tubulin that have essential roles segregating chromosomes during cell division, organizing the cytoplasm, establishing cellular polarity, and more. These fundamental activities depend critically on dynamic instability, the stochastic switching of microtubules between phases of growth and rapid shrinking. Dynamic instability is itself a consequence of αβ-tubulin GTPase activity and how it affects interactions between αβ-tubulin in the lattice and at the microtubule end. Although predictive molecular understanding of catastrophe remains elusive, the broad outlines of an understanding have been established. Unpolymerized, GTP-bound αβ-tubulin subunits readily associate at the growing tips of the MTs. Once they are incorporated into the lattice, αβ-tubulin GTPase activity is accelerated. The assembly dependence of GTPase activity results in a "stabilizing cap" of GTP-bound αβ-tubulin near the ends of the growing microtubules. Loss of this stabilizing cap triggers catastrophe, the switch from growth to rapid shrinking, because it exposes the more labile GDP-bound microtubule lattice. And, regaining this GTP cap results in microtubule rescue, the switch from shrinking to growing phase. In Chapter 1, I discuss literature background for microtubule dynamics. In Chapter 2, I present a study in which I explored the molecular mechanism behind microtubule catastrophe using computer simulations. By incorporating various candidates for "missing biochemistry" into the computational models, I discovered that lateral long-range interdimer interactions are crucial in correctly predicting catastrophe frequency as a function of the soluble tubulin concentration. In Chapter 3, I discuss a study that directly measures the αβ-tubulin association and dissociation at the microtubule end. We found that the kinetic on-rate of tubulin dimer to a growing microtubule end may be much smaller than previously thought. Next, in chapter 4, I present studies that explore various factors that affect microtubule shrinking and rescue. Finally, in chapter 5, I will summarize my work on these projects and discuss future directions and preliminary results looking to better understand microtubule dynamics.Item An Isolated Clasp TOG Domain Suppresses Microtubule Catastrophe and Promotes Rescue(2019-04-12) Majumdar, Shreoshi; Zhang, Xuewu; Rice, Luke M.; Yu, Hongtao; Tu, BenjaminMicrotubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the CLASP family that reduce catastrophe and/or increase rescue also contain arrayed TOGs. How CLASP TOGs contribute to activity is poorly understood. Using S. cerevisiae Stu1 as a model CLASP, I report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. To begin with, I introduce microtubules, their dynamics and regulatory proteins in Chapter 1. In Chapter 2, I discuss how the two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. I also explore the structure of TOG2 and how it reveals a CLASP-specific residue that likely dictates distinctive tubulin-binding properties. Next, in Chapter 3, I study how, contrary to the expectation that TOGs must work in arrays, the isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function. Finally, in Chapter 4, I will summarize my work and provide insight into future directions.Item Protein Composition and Subcellular Localization of the De Novo Lipogenic Metabolon(2016-04-18) McKean, William Bennion, Jr.; DeBose-Boyd, Russell A.; Horton, Jay D.; Russell, David W.; Uyeda, KosakuFatty acids are the major components of triglycerides, phospholipids, and sphingolipids. Production of palmitate, the most abundant saturated fatty acid, involves the stepwise actions of three enzymes: ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase. Canonically each enzyme catalyzes discrete reactions, and it is thought that they localize diffusely in cellular cytoplasm separate from one another. If true, transfer of metabolic intermediates must occur through passive diffusion from one lipogenic enzyme to another. Such a model proposes an extremely inefficient and potentially hazardous method of palmitate production. We demonstrated that two related proteins - designated MIG12 and Spot 14 - modulate fatty acid synthesis and triglyceride production by regulating the polymerization and activity of acetyl-CoA carboxylase. To better characterize the relationship between these three proteins, biochemical properties of purified recombinant MIG12, Spot 14, and MIG12:Spot 14 heterodimer were assayed in combination with acetyl-CoA carboxylase. We found that Spot 14 abrogates the ability of MIG12 to polymerize and activate acetyl-CoA carboxylase. Co-immunoprecipitation studies using Spot 14 in rat liver revealed Spot 14 exists in a complex with fatty acid synthase and acetyl-CoA carboxylase. MIG12 and Spot 14 co-immunoprecipitation also revealed that ATP citrate lyase was in association with the complex, suggesting that these proteins can function as scaffolds for the three enzymes required for palmitate synthesis. Studies of the subcellular localization of these lipogenic proteins corroborated a functional interaction between these proteins. Confocal images of MIG12 and acetyl-CoA carboxylase in primary hepatocytes show filamentous structures that are immunofluorescent along junctions between the endoplasmic reticulum and mitochondria. Under high carbohydrate dietary conditions in which lipogenesis is stimulated, these structures expand to include fatty acid synthase, ATP citrate lyase, and Spot 14. They also co-localize around lipid droplets - storage organelles for excess triglycerides. Finally, the structural integrity of this lipogenic complex is shown to require microtubules. Blockade of microtubule formation inhibits proper formation of acetyl-CoA carboxylase structure and decreases total fatty acid synthesis in cells. Combined, these findings support the existence of a functional metabolon complex which facilitates the efficient channeling of fatty acid synthesis intermediates through an enzyme cascade that results in the production of palmitate at functionally relevant locations within the cell.Item Recombinant αβ-Tubulin and a Simple Computational Model Shed Light on the Molecular Mechanisms of Microtubule Dynamics(2015-02-06) Piedra, Felipe-Andrés; Yu, Hongtao; Ranganathan, Rama; Ross, Elliott M.; Rice, Luke M.Microtubules (MTs) are essential to all eukaryotic organisms. They help segregate chromosomes and organize the cytoplasm. MTs are hollow barrels of the protein αβ-tubulin that exhibit a non-equilibrium behavior called dynamic instability: the stochastic switching of single polymers from a state of gradual growth to one of rapid disassembly. Dynamic instability underlies the MT cytoskeleton's rapid reorganizability and enables its diversity of functions. MTs can be reconstituted from purified αβ-tubulin and have been studied in vitro for over 40 years. Over this time, huge strides have been made in the development of an understanding of dynamic instability. Nevertheless, the mechanistic basis of important phenomena like GTP-dependent assembly and GTP hydrolysis-induced conformational change and catastrophe (the switch from growing to shrinking) remain controversial or unexplained. In Chapter 2, I discuss a study in which we used a computational model to investigate the consequences of a new way of thinking about the effect of nucleotide-state on αβ-tubulin and MT assembly. Our results suggest that GDP exposure on the MT plus-end can frustrate elongation and lead to catastrophe. We therefore predicted that GDP to GTP exchange on the MT plus-end might reduce the frequency of catastrophe. We tested our prediction by analyzing the effects of a mutant αβ-tubulin and a GTP analog designed to increase the rate of terminal nucleotide exchange on MT dynamics in vitro. Our experimental results support the results from our model. Thus, we believe that GDP exposure on the MT plus-end increases the likelihood of catastrophe, and can be countered by GDP to GTP exchange. In Chapter 3, I discuss a comparison of yeast and porcine MT dynamics in vitro. My measurements reveal striking differences between yeast and mammalian MT dynamics, and provide new constraints for models of MT dynamics. I conclude my thesis in Chapter 4 with my view of what my work means, what remains to be done and what paths my work has opened for further exploration.Item Safeguard of Mitosis: The Spindle Checkpoint(2016-11-18) Ji, Zhejian; Chen, Zhijian J.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoIn mitosis, the kinetochore-microtubule attachment is under surveillance by the spindle checkpoint to ensure the fidelity of chromosome segregation. Defects in the checkpoint could lead to aneuploidy, which has been implicated in cancers, birth defects, and other human diseases. In presence of kinetochores that are not attached or improperly attached to microtubules, the checkpoint signals to assemble the mitotic checkpoint complex (MCC), which consists of BubR1-Bub3, Mad2, and Cdc20. The diffusible MCC inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome bound to its co-activator Cdc20 (APC/C-Cdc20) to arrest cells in mitosis. Nevertheless, it remains unknown how the checkpoint monitors the status of the kinetochore-microtubule attachment. Neither is clear how MCC is assembled in an active checkpoint signaling. My graduate work has answered these two questions by revealing the critical functions of a checkpoint kinase, monopolar spindle 1 (Mps1), in both attachment sensing and checkpoint signaling. Of kinetochore proteins, the KMN network acts as both a critical microtubule receptor and a signaling platform for the spindle checkpoint. The human KMN contains the kinetochore null 1 complex (Knl1C), the minichromosome instability 12 complex (Mis12C), and the nuclear division cycle 80 complex (Ndc80C). In my first project, I have shown that the non-kinase domain of Mps1 directly binds to Ndc80C through two independent interactions. Both interactions involve the microtubule-binding surfaces of Ndc80C and are directly inhibited by microtubules. Elimination of one such interaction in human cells causes checkpoint defects expected from a failure in detecting unattached kinetochores. This competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for unattached kinetochore detection. The next question is how the kinetochore-associated Mps1 kinase rules the checkpoint signaling. At kinetochore, Mps1 phosphorylates the scaffolding protein Knl1. Phosphorylated Knl1 (pKnl1) recruits checkpoint complexes budding uninhibited by benomyl 1-3 (Bub1-Bub3) and Bub1-related protein in complex with Bub3 (BubR1-Bub3) to kinetochores. My following work has demonstrated that Mps1 promotes the inhibition of APC/CCdc20 by MCC components in vitro through phosphorylating Bub1 and mitosis arrest deficiency 1 (Mad1). Phosphorylated Bub1 (pBub1) binds with Mad1-Mad2. Phosphorylated Mad1 (pMad1) directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through a pKnl1-pBub1-pMad1 phosphorylation cascade, in which phosphorylation of upstream components enables binding of downstream ones. We propose that this sequential multi-target phosphorylation cascade allows Mps1 to amplify checkpoint signals and makes the checkpoint highly responsive to Mps1, which itself is regulated by kinetochore-microtubule attachment. Taken together, my graduate work has solved two long-standing questions in spindle checkpoint regulation. Accordingly, Mps1 recognizes the unattached kinetochores via its non-kinase domain, while activates the checkpoint signaling through its kinase activity. The dual function of Mps1 couples checkpoint activation with unattached kinetochore detection, making checkpoint under the control of kinetochore-microtubule attachment.Item Structural Characterization and Chemical Inhibition of the ARNT/TACC3 Complex(2015-04-13) Guo, Yirui; Rosenbaum, Daniel M.; Roth, Michael G.; Rice, Luke M.; Gardner, Kevin H.My research project focuses on mechanistic studies of a new group of transcriptional coactivators (coiled-coil coactivators: TACC3, TRIP230, CoCoA), involved in cancer development and progression. Normally, these coactivators play an essential role in the HIF hypoxia response, directly interacting with the ARNT subunit of HIF in a novel and promoter-specific way. However, misregulation by overexpression or activating fusions (for example, FGFR-TACC3) is sufficient for transformation and associated with the development of glioblastoma, renal cell carcinoma and other cancers. In light of this connection between coiled-coil coactivators (CCCs) and HIF signaling, tools that inhibit HIF/CCC complex formation might present opportunities to interrogate the linkage between different CCC-containing pathways and may offer a novel route to blocking cancer formation and progression. As a member of a new group of transcription coactivators, knowing how TACC3 interacts with ARNT is critical in understanding the general role of CCCs in HIF-dependent transcription machinery. In the first half of my study, I characterized the ARNT/TACC3 complex with various biophysical and biochemical methods including solution NMR, X-ray crystallography, circular dichroism, luminescence proximity and numerous cell-based assays. A 3.15 Å ARNT/TACC3 crystal structure was solved by molecular replacement, revealing details of this protein complex and providing a structural funcation for coactivator recruitment in HIF signaling pathway. The second half of this study focuses on the search for ARNT/TACC3 inhibitors with in vitro screens to regulate ARNT/CCCs activity in a rapid and flexible way. From a fragment-based NMR screen, I identified small molecules that specifically bound within the second PER-ARNT-SIM (PAS) domain of ARNT and perturb its interaction with TACC3. However, these small molecules have drawbacks, such as low potency or unclear modes of action. To identify higher potency small molecules targeting ARNT/TACC3 complexes, I developed an AlphaScreen-based high throughput screen. Hopefully the discovery of artificial ligands with known mode-of-action that inhibit this typically "undruggable" protein complex will provide new perspectives in small molecule inhibitor development, and also serve as a very useful tool in cell biology for studying pathways utilizing ARNT/TACC3.Item A TOG:αβ-Tubulin Complex Structure Reveals Conformation-Based Mechanisms for a Microtubule Polymerase(2012-12-04) Ayaz, Pelin 1983-; Yu, Hongtao; Albanesi, Joseph P.; Rosen, Michael K.; Rice, Luke M.Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use alpha/beta-tubulin-interacting TOG (tumor overexpressed gene) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, but how they do so has remained unclear. In this study, we first introduce the polymerization blocked mutants of alpha/beta-tubulins that we developed as unique tools for biochemical studies of alpha/beta-tubulins to avoid the difficulties that has arisen from the self-assembly tendency of tubulins, then we report the structure of the TOG1 domain from Stu2p bound to the plus end polymerization blocked yeast alpha/beta-tubulin we created to facilitate crystallization. Our structure and further biochemical characterizations of the TOG1:alpha/beta-tubulin complex showed that TOG1 binds alpha/beta-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a “curved” conformation of alpha/beta-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with alpha/beta-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, and how they selectively recognize the growing end of the microtubule.