Steven Howell

Structure of mammalian AMPK and its regulation by ADP

The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.

Structure and catalytic mechanism of the human histone methyltransferase SET7/9

Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein–dynactin pathway by interacting with these complexes

Genetic analysis in Drosophila suggests that Bicaudal‐D functions in an essential microtubule‐based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal‐D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co‐localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein‐tagged forms of BICD2 verify its intracellular distribution and co‐localization with dynactin, and indicate that the C‐terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N‐terminal domain of BICD2 disrupts minus‐end‐directed organelle distribution and this portion of BICD2 co‐precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2–dynactin–dynein co‐localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein–dynactin interaction on the surface of membranous organelles, by associating with these complexes.

KSHV vFLIP binds to IKK-γ to activate IKK

When expressed in heterologous cells, the viral FLIP protein (vFLIP) of Kaposis-sarcoma-associated herpesvirus (KSHV) has been reported both to block Fas-mediated apoptosis and to activate the NF-κB activation pathway by interaction with IκB kinase (IKK). In a yeast-two-hybrid screen, we identified IKKγ as an interacting partner of vFLIP. We expressed fragments of IKKγ in mammalian cells and bacteria, and identified the central CCR3/4 (amino acids 150-272) as the vFLIP binding region. To investigate the proteins interacting with vFLIP in a KSHV-infected primary effusion lymphoma (PEL) cell line, we immunoprecipitated vFLIP and identified four associated proteins by mass spectrometry: IKK components IKKα, β and γ, and the chaperone, Hsp90. Using gel filtration chromatography, we demonstrated that a single population of vFLIP in the cytoplasm of PEL cells co-eluted and co-precipitated with an activated IKK complex. An inhibitor of Hsp90, geldanamycin, inhibited IKKs kinase activity induced by vFLIP and killed PEL cells, suggesting that vFLIP activation of IKK contributes to PEL cell survival.

Crystal Structure and Functional Analysis of the Histone Methyltransferase Set7/9

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.

Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach

We investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two‐dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up‐regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)‐8, osteopontin, monocyte chemotactic protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), regulated on activation, normal, T‐cell expressed and secreted (RANTES), MIP‐1β, MIP‐3α, myeloid progenitor inhibitory factor‐1 (MPIF‐1), pulmonary and activation regulated chemokine (PARC), growth regulated gene‐β (GRO‐β), GRO‐γ, MCP‐2, I‐309, and the T helper 2 (Th2) and eosinophil‐attracting chemokine, eotaxin. Other genes involved in cell migration which were up‐regulated included the matrix metalloproteinase MMP‐9, vascular endothelial growth factor (VEGF) and its receptor Flk‐1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule‐1 (VCAM‐1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL‐1β (showing a 433‐fold induction), IL‐2 and tumour necrosis factor‐α (TNF‐α), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL‐1β was again shown to be strongly up‐regulated. The enzyme manganese superoxide dismutase was also found to be strongly up‐regulated; this enzyme was found to be macrophage‐, rather than M. tuberculosis, derived. The heat‐shock protein hsp27 was found to be down‐regulated following infection. We also identified a mycobacterial protein, the product of the atpD gene (thought to be involved in the regulation of cytoplasmic pH) in the infected macrophage extracts.

A Single Malaria Merozoite Serine Protease Mediates Shedding of Multiple Surface Proteins by Juxtamembrane Cleavage

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actin-independent process.

14-3-3 IS PHOSPHORYLATED BY CASEIN KINASE I ON RESIDUE 233 : PHOSPHORYLATION AT THIS SITE IN VIVO REGULATES RAF/14-3-3 INTERACTION

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 ζ on Thr-233. This protein kinase has been identified as casein kinase Iα (CKIα) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 τ possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 ζ is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 ζ associates with the regulatory domain of c-Raf. We have now shown thatin vivo phosphorylation of 14-3-3 ζ at the CKIα site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.

The merozoite surface protein 6 gene codes for a 36 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex.

A complex of non-covalently bound polypeptides is located on the surface of the merozoite form of the human malaria parasite Plasmodium falciparum. Four of these polypeptides are derived by proteolytic processing of the merozoite surface protein 1 (MSP-1) precursor. Two components, a 22 and a 36 kDa polypeptide are not derived from MSP-1. The N-terminal sequence of the 36 kDa polypeptide has been determined, the corresponding gene cloned, and the protein characterised. The 36 kDa protein consists of 211 amino acids and is derived from a larger precursor of 371 amino acids. The precursor merozoite surface protein 6 (MSP-6) has been designated, and the 36 kDa protein, MSP-6(36). Mass spectrometric analysis of peptides released from the polypeptide by tryptic digestion confirmed that the gene identified codes for MSP-6(36). Antibodies were produced to a recombinant protein containing the C-terminal 45 amino acid residues of MSP-6(36). In immunofluorescence studies these antibodies bound to antigen at the parasite surface or in the parasitophorous vacuole within schizonts, with a pattern indistinguishable from that of antibodies to MSP-1. MSP-6(36) was present in the MSP-1 complex immunoprecipitated from the supernatant of in vitro parasite cultures, but was also immunoprecipitated from this supernatant in a form not bound to MSP-1. Examination of the MSP-6 gene in three parasite lines detected no sequence variation. The sequence of MSP-6(36) is related to that of the previously described merozoite surface protein 3 (MSP-3). The MSP-6(36) amino acid sequence has 50% identity and 85% similarity with the C-terminal region of MSP-3. The proteins share a specific sequence pattern (ILGWEFGGG-[AV]-P) and a glutamic acid-rich region. The remainder of MSP-6 and MSP-3 are unrelated, except at the N-terminus. Both MSP-6(36) and MSP-3 are partially associated with the parasite surface and partially released as soluble proteins on merozoite release. MSP-6(36) is a hydrophilic negatively charged polypeptide, but there are two clusters of hydrophobic amino acids at the C-terminus, located in two amphipathic helical structures identified from secondary structure predictions. It was suggested that this 35 residue C-terminal region may be involved in MSP-6(36) binding to MSP-1 or other molecules; alternatively, based on the secondary structure and coil formation predictions, the region may form an intramolecular anti-parallel coiled-coil structure.

Proteomic Analysis of Cleavage Events Reveals a Dynamic Two-step Mechanism for Proteolysis of a Key Parasite Adhesive Complex

The transmembrane micronemal protein MIC2 and its partner M2AP comprise an adhesive complex that is required for rapid invasion of host cells by the obligate intracellular parasite Toxoplasma gondii. Recent studies have shown that the MIC2/M2AP complex undergoes extensive proteolytic processing on the parasite surface during invasion, including primary processing of M2AP by unknown proteases and proteolytic shedding of the complex by an anonymous protease called MPP1. While it was shown that MPP1-mediated cleavage is necessary for efficient invasion, it remained unclear whether the adhesive complex was liberated by juxtamembrane or intramembrane proteolysis. Here, using a three-phase strategy of assigning cleavage sites based on intact matrix-assisted laser desorption/ionization mass followed by confirmation by enzymatic digestion and inhibitor profiling, we demonstrate that M2AP is processed by two parasite-derived proteases called MPP2 and MPP3. We also define the substrate repertoire of MPP2 by two-dimensional differential gel electrophoresis using fluorescent tags. Finally, we use complementary mass spectrometric techniques to unequivocally show that MIC2 is shed by intramembrane cleavage within its anchoring domain. Based on the properties of this cleavage site, we conclude that the sheddase, MPP1, is likely a multipass membrane protease of the Rhomboid family. Our data support a novel two-step proteolysis model that includes primary processing of the MIC2/M2AP complex followed by secondary cleavage to shed the complex from the parasite surface during the final steps of invasion.