Tania A. Baker - US grants

Phage Mu transposes at an extraordinarily high frequency and many fundamental studies of transposition have been done using Mu. It is now clear that key aspects of this recombination mechanism are shared by Mu and many other transposable elements that invade prokaryotic and eukaryotic organisms. The long term goal of this project is to understand the molecular mechanism of Mu transposition. It has recently become clear that Mu transposase is a member of a structurally related family of proteins that includes many transposases and the retroviral integrases. These proteins catalyze recombination as multimeric complexes bound to DNA. Understanding of the structural organization of a transposase-DNA complex in most advanced for the Mu transposase. The active form of Mu transposase is a homotetramer bound simultaneously to the three segments of DNA that participate in recombination. The specific goals of this proposal are aimed at providing a thorough description of how the DNA molecules are arranged within this tetrameric protein complex and how the complex is assembled. We will use protein- DNA and protein-protein cross-linking to map the regions of Mu transposase that interact with the DNA at the recombination sites and provide the protein-protein surfaces that hold the tetramer together. Assembly of the active transposase tetramer absolutely depends on binding of the protein to specific DNA sites; the structural basis of this DNA-dependence will be investigated using multiple probes for changes in protein conformation. Genetic and biochemical experiments to address how an appropriate DNA target site is selected during recombination are also proposed. This analysis of the mechanisms underlying the assembly and activation of the protein-DNA complexes involved in transposition is likely to provide insights into the mechanisms that control transcription and replication as well. The impact of transposition on human health is immense. The rapid spread of antibiotic resistance genes is largely a result of transposable elements moving throughout bacterial populations. Furthermore, retroviruses, including HIV, integrate into the host chromosome via a mechanism nearly identical to transposition. A related recombination reaction is also responsible for assembly of the immunoglobulin and T-cell receptor genes during development of the vertebrate immune system. Understanding the molecular mechanism of this important class of genetic recombination should assist the future design or discovery of agents that may prevent the undesirable consequences of transposition.

9357322 Baker The bacterial virus Mu i a member of a diverse group of genetic elements that promote insertion of their own DNA into the genome of a host cell by a specialized type of genetic recombination known as DNA transposition. Many elements, including retroviruses, utilize this mechanism of integration, allowing them to insert at nearly random sites in the host DNA. The long term goal of this project is to understand the molecular mechanism of transposition. The extraordinary efficiency of Mu transposition, and the nonpathogenic nature of the virus, make Mu an ideal system for mechanistic studies. Recently, it has become clear that the active form of the Mu transposase is a tetramer of the protein bound to the paired ends of the element DNA. Current knowledge of the chemical mechanism of transposition based on work principally from the Mu and HIV systems make it very attractive to consider that the protein is activated by tetramer assembly and that the active center of the protein may lie at constructed tetramer interface. The experiments proposed here will locate and dissect the regions of the transposase involved in catalysis and in tetramer contacts. The roles of individual monomers within the active tetramer will also be investigated. %%% Transposition is the recombination reaction by which a genetic element is moved from one DNA to another. Studying this important process will lead to better understanding of HIV DNA integration, as well as other complex DNA-protein interactions such as replication, transcription and RNA processing. ***

Higher-order protein-DNA complexes both orchestrate and catalyze many of life's most central processes. Genetic recombination and DMAprotection, processes essential for maintaining the integrity of the genome for future generations, are especially rich in their use of protein-DNA superstructures. A key feature of these complexes is that they are often exceedingly stable, such that ATP-dependent protein-unfolding enzymes and proteases may be required to remodel, dismantle, destroy or recycle the component proteins. The focus of this project is to understand how protein complexes are recognized for remodeling or destruction by proteins of the Clp/Hsp100 ATPase family. Clp/Hsp100 proteins are a subfamily of the AAA+ enzymes that use ATP-hydrolysis to perform mechanical work on their substrates. The first specific goal is to understand how the protein-unfolding enzyme CIpX recognizes the protein-DNA complex that promotes DNA transposition of phage Mu. Experiments to elucidate the peptide signals within the transposase responsible for recognition are proposed. Furthermore, we will test a model in which asymmetric features of the complex guide the unfolding activity of CIpX to one specific transposase subunit, and thereby generate a new -less stable-complex with a unique architecture. The second goal is to elucidate how the DNA- protection protein Dps is recognized by CIpX and how this recognition is tuned to changing environmental conditions. The role of peptide signals, adaptor proteins and DNA in recognition of Dps will be investigated. Finally, proteomic experiments designed to achieve a global view of the role of ATP-dependent protein unfoldases/proteases are proposed. These experiments will give a proteome-wide view of the roles of specific adaptor proteins and peptide binding domains in substrate choice by AAA+ enzymes. Cellular mechanisms that protect the genome from damage, and promote the faithful repair of damaged DNA are critical to health and survival, as dramatically demonstrated by the numerous cancer syndromes associated with the genetic disruption of these cellular processes. Protein remodeling and destruction byAAA+ enzymes is a critical yet poorly understood aspect of the strategies used by cells to both interpret and protect their genomes.

DESCRIPTION (provided by applicant): This proposal is for continuing support of the pre-doctoral training grant to the Department of Biology at the Massachusetts Institute of Technology (MIT). This training grant (coming up on year 34) continues to be the most important source of support for graduate students studying biological science at MIT. The mission of this Graduate Program is to train the next generation of biological/biomedical scientists, many of whom will be innovators and leaders in research and education. Specifically, in this training program we strive to educate our students: to deeply understand the fundamental underlying principles of modern biology including genetics, biochemistry, cell biology, molecular biology and quantitative data analysis;to be ethical decision makers;to face the rapidly changing modern scientific landscape;to become creative, effective, rigorous researchers;and to become excellent teachers and mentors of younger students. We seek out, recruit and train excellent students from majority, underrepresented minority, and disadvantaged populations, and help them initiate successful research careers. A key feature of our training program is an intensive, focused curriculum required of all first semester students. During this semester, students work together in courses taught by dedicated faculty in lecture and discussion-style to master a fundamental toolbox of approaches that are the underpinning of all modern molecular biological science. New features of the program include a required course in quantitative and computational biology and a writing tutorial on the preparation of research proposals. The training program ensures that students are exposed to all research groups in the Department before choosing their three lab rotations, ensuring that they are well prepared to make the critical choice of a thesis lab. Responsible conduct in research is taught in three phases, including an intense mini-course for 2nd year students. The progress and completion of thesis research is carefully monitored by regular thesis committees meetings and by the Graduate Committee. Our students perform research of outstanding quality and most students go on to careers in biomedical research. Many of our former trainees are now leaders in their chosen fields. RELEVANCE: Key to Combating the complex problems plaguing human health are scientists rigorously trained in the fundamental aspects of molecular and cellular biology, in ethical and humane decision-making, and exposed to the problems of modern medicine. Our program strives for excellence in all these areas. Our program is the major source of graduate students to the Koch Institute of Integrative Cancer Research and many other laboratories whose research has a direct impact on human health and disease.

DESCRIPTION (provided by applicant): AAA+ proteases are responsible for intracellular protein degradation in all domains of life and controlled ATP-dependent protein degradation and regulated gene expression are partners in defining the proteome. Furthermore, the same enzymatic machinery used for degradation is critical for disassembling protein complexes and antagonizing protein aggregation. As both degradation and disassembly are inherently destructive processes and the efficiency of substrate breakdown is often made at the level of substrate choice, it is imperative to understand the molecular principles guiding substrate selection by these ATP-driven, protein-destruction machines. This proposal focuses on three areas of substrate selection of the bacterial AAA+ proteases ClpXP, ClpAP and Lon. Aim 1 concentrates on elucidating the design principles and molecular contacts used by two specific classes of ClpX substrate-recognition signals. As part of this aim, we test the hyphothesis that some signals are "designed" such that they are preferentially recognized in the context of multi-protein complexes. We also propose to solve the structure of newly-discovered ClpX N-domain-interacting motifs bound to the N-domain to further understand the mechanistic basis of ClpX recognition. Aim 2 dissects how Lon and ClpAP proteases and the E. coli sHsps (IbpA and IbpB) interact, tests models for how these interactions impact protein quality-control pathways. We are excited to explore the functional consequences of this newly-discovered intersection between the aggregation-prevention (sHsp) and protein-destruction (protease) arms of the protein quality-control network. The final aim begins to address mechanisms for control of protein degradation in response to oxidative stress and oxidative damage. Two proteins are chosen for initial studies: the B. subtilis peroxide-sensing transcription factor, PerR and the E. coli mini-ferritin, Dps. Preliminary data indicate that PerR is specifically subject to accelerated degradation by LonA protease when its histidine active center is irreversibly oxidized. In contrast to PerR, Dps degradation is inhibited by peroxide. We propose to isolate factors responsible for these examples of environmentally-controlled regulation. Successful completion of these aims will provide substainal new insights into protease recognition and may uncover new paradigms for contol of the proteome. PUBLIC HEALTH RELEVANCE: AAA+ proteases are virulence factors in many bacteria including major human pathogens. Elucidating rules of substrate recognition will be key to identifying unstable virulence-associated proteins and understanding how their stabilization leads to pathogenesis. Analysis of the connection between proteases and the sHsps (which are proteins that fight aggregation), and elucidating how proteases recognize oxidatively damaged proteins, holds promise for uncovering new cellular mechanisms used to fight age- and reactive oxygen-associated protein toxicity.

Heme is the oxygen-binding ligand of hemoglobin and is an essential cofactor or sensor element in many proteins. Heme production must be tightly controlled to adequately supply these functions but to avoid overproduction, as accumulation of free heme and heme precursors is toxic. The first committed step in heme biosynthesis is the condensation of glycine and succinyl-CoA to yield 5-aminolevulinic acid (ALA). This reaction is catalyzed by ALA synthase (ALAS), which uses pyridoxal 5?-phosphate (PLP, the active form of vitamin B6) as an essential cofactor. In animals, there are two differentially expressed ALAS isoforms. ALAS1 is present in most cells, whereas ALAS2 is an erythroid-specific enzyme that is dramatically upregulated during red cell development. In humans, mutations in ALAS2 cause two diseases: (1) X-linked sideroblastic anemia (XLSA) when enzyme activity is too low to support healthy levels of heme production and erythropoiesis and (2) Erythroid X-linked protoporphyria (XLPP), from gain-of-function ALAS2 mutations that overproduce ALA, causing build up of toxic heme biosynthetic intermediates. The life cycle of ALAS is tightly regulated at steps including mitochondrial import and protein turnover. Both these steps are feedback controlled by heme-binding. Enzyme activity (and/or stability) is also regulated and these processes are affected by interaction with other enzymes, including Lon protease, succinyl-CoA synthetase (SCS), and perhaps ferrochelatase (FECH), the final two also critical enzymes in heme synthesis. Importantly, we recently discovered that ALAS activity is also dramatically stimulated by mitochondrial ClpX (mtClpX), a member of the AAA+ family of protein unfoldases. The mtClpX energy-dependent unfoldase accelerates incorporation of PLP into ALAS and CLPX depletion causes anemia in vertebrates. We also solved structures of both PLP-free ALAS (from yeast) and the active PLP-bound enzyme, which illuminates the conformational changes coupled to PLP incorporation and provides important information for understanding mtClpX-promoted loading of PLP. These structures also provide the first observation of the eukaryotic-specific regulatory C-terminal domain of the enzyme. This domain structure suggests testable mechanisms to explain the XLPP mutations and contains the binding site for SCS, which we will further study. Continuing to investigate how mtClpX physically interacts with ALAS and to test models for the mechanism of PLP-loading holds promise for uncovering a link between mtClpX-ALAS2 interactions and some classes of XLSA alleles. In another recent, exciting breakthrough, our collaborators discovered a dominant human CLPX mutation that appears to hyperactivate ALAS, leading to mtClpX-linked erythropoietic protoporphyria (EPP). The mechanistic basis of this disease will be scrutinized at the molecular, structural and cellular level. Thus, by probing the complex mechanisms that control ALAS enzymes we will elucidate new molecular means of regulation. We believe that this work, in turn, will inspire novel therapeutic strategies for combating the debilitating illnesses caused by misregulated ALAS.