Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance mechanism that requires upframeshift protein 1 (UPF1). This study demonstrates that human UPF1 exerts protective effects in a rat paralysis model based on the amyotrophic lateral sclerosis (ALS)-associated protein, TDP-43 (transactive response DNA-binding protein 43 kDa). An adeno-associated virus vector (AAV9) was used to express TDP-43 throughout the spinal cord of rats, inducing reproducible limb paralysis, to recapitulate the paralysis in ALS. We selected UPF1 for therapeutic testing based on a genetic screen in yeast. The expression of human TDP-43 or human UPF1 in the spinal cord was titrated to less than twofold over the respective endogenous level. AAV9 human mycUPF1 clearly improved overall motor scores in rats also expressing TDP-43. The gene therapy effect of mycUPF1 was specific and reproducible compared with groups receiving either empty vector or green fluorescent protein vector controls. The gene therapy maintained forelimb motor function in rats that would otherwise become quadriplegic. This work helps validate UPF1 as a novel therapeutic for ALS and other TDP-43-related diseases and may implicate UPF1 and NMD involvement in the underlying disease mechanisms.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aß42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Interaction of a peptidomimetic aminimide inhibitor with elastase.

The crystal structure of an aminimide analog of a dipeptide inhibitor of porcine pancreatic elastase bound to its target serine protease has been solved. The peptidomimetic molecule binds in the same fashion as the class of dipeptides from which it was derived, making similar interactions with the subsites on the elastase surface. Because aminimides are readily synthesized from a wide variety of starting materials, they form the basis for a combinatorial chemistry approach to rational drug design.

A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids.

Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

On the origin of enzymatic species.

The diversity of enzyme catalytic function is remarkable, particularly when one considers that ancestral life forms must have started with a much smaller ensemble of proteins. In this article, we discuss the evolution of the mandelate pathway in pseudomonads as an example of how catalytic diversity may have evolved. We suggest that existing enzymes that catalyse the chemistry needed to accomplish a transformation were recruited, followed by the evolution of specific binding.

What makes a binding site a binding site?

Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.

Proteins in organic solvents.

Catalysis in organic solvents and the mapping of protein surfaces using multiple solvent crystal structures are two rapidly developing areas of research. Recent advances include the study of protein folding and stability in different solvents, and the demonstration that it is possible to qualitatively rank the affinities of protein binding sites for a given organic solvent using the multiple solvent crystal structures method.

Locating and characterizing binding sites on proteins.

This review article begins with a discussion of fundamental differences between substrates and inhibitors, and some of the assumptions and goals underlying the design of a new ligand to a target protein. An overview is given of the methods currently used to locate and characterize ligand binding sites on protein surfaces, with focus on a novel approach: multiple solvent crystal structures (MSCS). In this method, the X-ray crystal structure of the target protein is solved in a variety of organic solvents. Each type of solvent molecule serves as a probe for complementary binding sites on the protein. The probe distribution on the protein surface allows the location of binding sites and the characterization of the potential ligand interactions within these sites. General aspects of the application of the MSCS method to porcine pancreatic elastase is discussed, and comparison of the results with those from X-ray crystal structures of elastase/inhibitor complexes is used to illustrate the potential of the method in aiding the process of rational drug design.

Observation of unstable species in enzyme-catalyzed transformations using protein crystallography.

Recent advances in rapid X-ray diffraction data collection methods, cryocrystallography, and other techniques have made it possible to visualize short-lived species in enzyme-catalyzed reactions directly at atomic resolution for a significant number of crystalline enzymes. The wide range of reaction types, intermediate lifetimes, and crystal characteristics means that different methods must be employed in each case, but there are enough examples now of successful structure determinations of normally unstable species to suggest guidelines for future investigations.

Preliminary X-ray data for aspartate aminotransferase from Escherichia coli.

Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221. Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees. There is one protein subunit of molecular weight 43,600 per asymmetric unit.

Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

Expression, crystallization and preliminary crystallographic analysis of human carbonyl reductase.

The cDNA of human placental carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase family of enzymes, was introduced into the plasmid vector pET-11a and the enzyme overexpressed in Escherichia coli. Recombinant carbonyl reductase was purified to homogeneity, characterized physically and kinetically, and crystallized for X-ray diffraction study. The recombinant protein was indistinguishable from human tissue carbonyl reductase (CR8.5 form) on the basis of partial sequence analysis, substrate specificity, susceptibility to inhibitors and immunochemical analysis. Similar to the tissue enzyme which which occurs in multiple molecular forms thought to arise from autocatalytic modification by 2-oxocarboxylic acids, a second form of the recombinant enzyme was generated under bacterial growth conditions producing high pyruvate concentrations. Purified recombinant protein, which corresponds to the smallest, most basic tissue form (CR8.5), was crystallized against 20% polyethyleneglycol 6000 in 25 mM 2-(N-morpholino)ethanesulfonic acid buffer (Mes) at pH 6.0 using the hanging drop method. Crystals of human carbonyl reductase diffract to better than 3.0 A, and the diffraction symmetry is consistent with a crystal that belongs to the tetragonal space group P4(1)(3)2(1)2 with unit cell dimensions of a = b = 55 A, c = 175 A, alpha = beta = gamma = 90.0. The asymmetric unit contains one molecule of 30.2 kDa.

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.

Structure of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesis.

Aspartate beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in an essential aspartic biosynthetic pathway found in bacteria, fungi and the higher plants. Mutations in the asd gene encoding for ASADH that produce an inactive enzyme are lethal, which suggests that ASADH may be an effective target for antibacterial, herbicidal and fungicidal agents. We have solved the crystal structure of the Escherichia coli enzyme to 2.5 A resolution using single isomorphous replacement and 3-fold non-crystallographic symmetry. Each monomer has an N-terminal nucleotide-binding domain and a dimerisation domain. The presence of an essential cysteine locates the active site in a cleft between the two domains. The functional dimer has the appearance of a butterfly, with the NADP-binding domains forming the wings and the dimerisation domain forming the body.A histidine residue is identified as a likely acid/base catalyst in the enzymic reaction. Other amino acids implicated in the enzymic activity by mutagenesis are found in the active site region and define the substrate binding pocket.

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus.

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure.

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity.

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.

Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.