Michael S. Cosgrove

Structure of a Sir2 enzyme bound to an acetylated p53 peptide

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide.

Mixed lineage leukemia: a structure–function perspective of the MLL1 protein

Several acute lymphoblastic and myelogenous leukemias are correlated with alterations in the human mixed lineage leukemia protein‐1 (MLL1) gene. MLL1 is a member of the evolutionarily conserved SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for the regulation of distinct groups of developmentally regulated genes in metazoans. Despite the important biological role of SET1 family enzymes and their involvement in human leukemias, relatively little is understood about how these enzymes work. Here we review several recent structural and biochemical studies that are beginning to shed light on the molecular mechanisms for the regulation of H3K4 methylation by the human MLL1 enzyme.

Histone proteomics and the epigenetic regulation of nucleosome mobility

Chromatin structure plays a vital role in the transmission of heritable gene expression patterns. The recent application of mass spectrometry to histone biology provides several striking insights into chromatin regulation. The continuing identification of new histone post-translational modifications is revolutionizing the ways in which we think about how access to genomic DNA is controlled. While post-translational modifications of the flexible histone tails continue to be an active area of investigation, the recent discovery of multiple modifications in the structured globular domains of histones provides new insights into how the nucleosome works. Recent experiments underscore the importance of a subgroup of these modifications: those that regulate histone–DNA interactions on the lateral surface of the nucleosome. This information highlights an emerging new paradigm in chromatin control, that of the epigenetic regulation of nucleosome mobility.

The LRS and SIN domains: two structurally equivalent but functionally distinct nucleosomal surfaces required for transcriptional silencing

ABSTRACT Genetic experiments have identified two structurally similar nucleosomal domains, SIN and LRS, required for transcriptional repression at genes regulated by the SWI/SNF chromatin remodeling complex or for heterochromatic gene silencing, respectively. Each of these domains consists of histone H3 and H4 L1 and L2 loops that form a DNA-binding surface at either superhelical location (SHL) ±2.5 (LRS) or SHL ±0.5 (SIN). Here we show that alterations in the LRS domain do not result in Sin− phenotypes, nor does disruption of the SIN domain lead to loss of ribosomal DNA heterochromatic gene silencing (Lrs− phenotype). Furthermore, whereas disruption of the SIN domain eliminates intramolecular folding of nucleosomal arrays in vitro, alterations in the LRS domain have no effect on chromatin folding in vitro. In contrast to these dissimilarities, we find that the SIN and LRS domains are both required for recruitment of Sir2p and Sir4p to telomeric and silent mating type loci, suggesting that both surfaces can contribute to heterochromatin formation. Our study shows that structurally similar nucleosomal surfaces provide distinct functionalities in vivo and in vitro.

Myosin5a Tail Associates Directly with Rab3A-containing Compartments in Neurons

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.

Biochemistry of the Mixed Lineage Leukemia 1 (MLL1) Protein and Targeted Therapies for Associated Leukemia

Mixed Lineage Leukemia constitutes a heterogeneous category of rare acute leukemias that are characterized by a mixed population of poorly differentiated lymphoid and myeloid progenitor cells. The mixed lineage leukemia (MLL1) gene, also known as HRX or ALL-1, is a frequent site of genetic rearrangements in infant acute leukemias and therapy-related malignancies (Daser and Rabbitts, 2005) and since its discovery (Djabali et al., 1992; Tkachuk et al., 1992; Ziemin-van der Poel et al., 1991), significant progress has been made in understanding its role in human biology and leukemogenesis (Liu et al., 2009; Slany, 2009). Chromosomal abnormalities involving the MLL1 gene include reciprocal chromosomal translocations, internal partial tandem duplications (PTD), and amplifications of unrearranged MLL1 (Dou and Hess, 2008). These chromosomal aberrations are associated with mechanistically distinct gain-of-function phenotypes that may be amenable to targeted therapeutic approaches. However, progress in this area has been impeded by a lack of understanding of the molecular details by which MLL1 translocations, amplifications and PTDs contribute to leukemogenesis. To date, more than 60 MLL1 fusion partners have been described (Krivtsov and Armstrong, 2007), and detailed genetic/biochemical studies have identified several functional domains within chimeric MLL1-fusion proteins that are essential for leukemic transformation (Daser and Rabbitts, 2005; Debernardi et al., 2002; Eguchi et al., 2004; Ernst et al., 2002; Krivtsov and Armstrong, 2007; Lavau et al., 2000; Liu et al., 2009; Luo et al., 2001; Mitterbauer-Hohendanner and Mannhalter, 2004; Mueller et al., 2009; Prasad et al., 1995). Although our understanding of the molecular pathology of MLL1associated leukemias remains incomplete, recent biochemical and structural information is contributing to an evolution of potential treatment strategies from a broadly-based chemotherapeutics approach towards therapies targeted to the underlying molecular pathogenesis of leukemia (Liedtke and Cleary, 2009). This chapter reviews recent advances in our efforts to develop novel MLL1-targeted therapies.