The mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, a key component of the Carma1/Bcl10/ MALT1 signalosome, is critical for NF-κB signaling in multiple contexts. MALT1 is thought to function as a scaffold and protease to promote signaling; however, the biochemical and structural basis of paracaspase action remains largely unknown. Here we report the 1.75-Å resolution crystal structure of the MALT1 paracaspase region, which contains the paracaspase domain and an ensuing Ig-like domain. The paracaspase and the Ig domains appear as a single folding unit and interact with each other through extensive van der Waals contacts and hydrogen bonds. The paracaspase domain adopts a fold that is nearly identical to that of classic caspases and homodimerizes similarly to form an active protease. Unlike caspases, the active and mature form of the paracaspase domain remains a single uncleaved polypeptide and specifically recognizes the bound peptide inhibitor Val-Arg-Pro-Arg. In particular, the carboxyl-terminal amino acid Arg of the inhibitor is coordinated by three highly conserved acidic residues. This structure serves as an important framework for deciphering the function and mechanism of paracaspases exemplified by MALT1.T he transcription factor NF-κB is a key constituent of all cell types and is activated by various receptors to regulate survival, proliferation, migration, and differentiation (1). In particular, NF-κB functions early in the development and maintenance of innate and adaptive immune systems and execution of the immune response. Although caspases, cysteine proteases that cleave substrate proteins after aspartate residues, are widely known as executioners of programmed cell death or apoptosis (2), a subset and related members also activate NF-κB to promote lymphocyte proliferation and inflammation. One such caspase-like family member, the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase, was identified through weak sequence homology to caspases (3) and was subsequently found to play an important role in lymphocyte activation (4) and disease progression in MALT lymphomas (5).Upon antigen receptor stimulation, the MALT1 paracaspase and Bcl10 assemble into the Carma1/Bcl10/MALT1 (CBM) signalosome to activate NF-κB in the adaptive immune system. Specifically during T-cell receptor signaling, the CBM signalosome is thought to oligomerize MALT1 and its associated ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) or TRAF2 (6, 7), which in turn facilitates K63-linked polyubiquitylation of multiple proteins including the regulatory γ-subunit of the IκB kinase (IKK) complex (6, 7), TRAF6 itself (7), Bcl10 (8), and MALT1 (9). Poly ubiquitylation of these proteins ultimately leads to the recruitment of transforming growth factor β-activated kinase 1 (TAK1), TAK1 binding protein (TAB), and the IKK complex to lipid rafts where the IKKβ-subunit is phosphorylated and activated. In the canonical NF-κB pathway, the activated IKK complex ph...

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The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors β-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis. Cancer Res; 70(11); 4422-32. ©2010 AACR.

Lipoic acid is the covalently attached cofactor of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzymebound lipoyl-AMP to the ⑀-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.Lipoic acid (6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid), a covalently bound cofactor, is essential for function of several key enzymes involved in oxidative metabolism in most prokaryotic and eukaryotic organisms (1). The lipoylated proteins include pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase, and the glycine cleavage system (2). In the reaction catalyzed by the lipoylating enzymes, i.e. lipoate-protein ligases (LPLs), 3 the free carboxyl group of lipoic acid is attached via an amide linkage to the ⑀-amino group of a specific lysine residue of the lipoate-accepting protein domains (termed the lipoyl domains) of these multienzyme complexes (3). The lipoamide arm protruding from a tight ␤-turn of the structure of the lipoyl domains shuttles reaction intermediates among different active sites of the multienzyme complexes (2). Although the general role of lipoic acid as the covalently attached coenzyme has been known for decades, the mechanisms by which lipoic acid is synthesized and becomes linked to its cognate proteins continue to be elucidated. In Escherichia coli two independent LPL enzymes modify lipoyl domains (4). The best characterized lipoylating enzyme is E. coli lipoate-protein ligase A (LplA). LplA utilizes exogenously supplied free lipoic acid to modify the specific lysine of the lipoyl domain. In the first step, LplA catalyzes synthesis...

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from the exocyst complex and appears well-suited for vesicle capture and fusion.

Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His 152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.Nitroalkanes are widely used in industry, because they are useful as intermediate compounds in chemical synthesis (1, 2). They are also synthesized by various organisms. Many antibiotics, e.g. chloramphenicol and azomycin, contain nitro groups, and many leguminous plants produce nitro toxins such as 3-nitro-1-propionic acid and 3-nitro-1-propanol (3). However, many nitroalkanes are expected to be toxic, and some have been shown to be carcinogenic (4 -10). For example, 2-nitropropane causes the formation of both 8-hydroxy-and 8-aminoguanine in the DNA and RNA (11). The enzymatic oxidation of nitroalkanes into less toxic species can therefore be exploited for use in bioremediation.2-Nitropropane dioxygenase (EC 1.13.11.32), one of the nitroalkaneoxidizing enzyme families, catalyzes oxidative denitrification of nitroalkanes to their corresponding carbonyl compounds and nitrites. To date, 2-nitropropane dioxygenase has been isolated from a fungus Neurospora crassa (12) and a yeast Williopsis mrakii (Hansenula mrakii) (13).The two enzymes have similar molecular masses of ϳ40 kDa, but their prosthetic groups are different. FMN and FAD are found in the N. crassa and W. mrakii (H. mrakii) enzymes, respectively (14, 15). The ncd-2 gene encoding for 2-nitropropane dioxygenase in N. crassa has been cloned and expressed in Escherichia coli (16). The heterologously expressed enzyme was found to be a homodimer containing 1 mol of non-covalently bound FMN per mole of subunit (16). A steady-state kinetic analysis showed that the preferred substrates for the enzyme are anionic nitronates as compared with neutral nitroalkanes and that the enzyme has broad substrate specificity that is independent of substrate size (16).It has been shown that 2-nitropropane dioxygenase operates through an oxidase-style catalytic mechanism, in which substrate oxidation occurs prior to and independently ...

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The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1-4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG's eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5-Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5-Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.I n eukaryotes, the transport of proteins and lipids among intracellular compartments is mediated by vesicular and tubular carriers under the direction of an elaborate protein machinery (1). Among the most complex and least well-characterized components of this machinery are the multisubunit tethering complexes (MTCs) (2). MTCs are thought to mediate the initial attachment (or tethering) between a trafficking vesicle and its target membrane through a constellation of interactions (3, 4). These may include binding of the MTC to activated Rab GTPases, coiled-coil proteins such as Golgins, vesicle coat proteins, SNAREs, Sec1/ Munc18 (SM) proteins, and/or membrane lipids. Elucidating the 3D structures of MTCs represents an important step toward a better understanding of their molecular functions.Four of the known MTCs-termed complexes associated with tethering containing helical rods (CATCHR) or quatrefoil complexes (2, 5)-contain subunits whose shared 3D structure implies a single evolutionary progenitor (6-16). These CATCHR-family MTCs include the Dsl1, Golgi-associated retrograde protein (GARP), exocyst, and conserved oligomeric Golgi (COG) complexes, and they contain three, four, eight, and eight subunits, respectively. Although X-ray or NMR structures have been reported for 14 of these 23 subunits, only one of the structures contains the full-length polypeptide (14). Perhaps more critically, only two subunit interactions-both wit...