Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia

Mild to moderate hyperhomocysteinemia is a risk factor for neurodegenerative diseases. Human studies suggest that homocysteine (Hcy) plays a role in brain damage, cognitive and memory decline. Numerous studies in recent years investigated the role of Hcy as a cause of brain damage. Hcy itself or folate and vitamin B12 deficiency can cause disturbed methylation and/or redox potentials, thus promoting calcium influx, amyloid and tau protein accumulation, apoptosis, and neuronal death. The Hcy effect may also be mediated by activating the N-methyl-D-aspartate receptor subtype. Numerous neurotoxic effects of Hcy can be blocked by folate, glutamate receptor antagonists, or various antioxidants. This review describes the most important mechanisms of Hcy neurotoxicity and pharmacological agents known to reverse Hcy effects.     

Control of Catalytic Cycle by a Pair of Analogous tRNA Modification Enzymes

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here, we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3′ position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor. TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinity to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed half-of-the-sites reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel with those between the two classes of aminoacyl-tRNA synthetases, which use distinct active site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.     

Crystal structure of rat liver betaine homocysteine s-methyltransferase reveals new oligomerization features and conformational changes upon substrate binding

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit. Moreover, the crystal structure of free rat liver BHMT clearly shows that Tyr160 is the fourth ligand coordinated to Zn, which is replaced by Hcy upon binding. Two residues essential for substrate recognition, Phe76 and Tyr77, are provided by a conformational change in a partially disordered loop (L2). The crucial role of these residues is highlighted by site-directed mutagenesis.     

Molecular characterization of porcine MMP19 and MMP23B genes and its association with immune traits

MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of the extracellular matrix. They are thought to play important roles in embryonic development, reproduction and tissue remodeling, as well as in cell proliferation, differentiation, migration, angiogenesis, apoptosis and host defense. However, they are poorly understood in pigs. Here, we obtained the full length coding region sequence and genomic sequence of the porcine MMP19 and MMP23B genes and analyzed their genomic structures. The deduced amino acid sequence shares similar precursor protein domains with human and mouse MMP19 and MMP23B protein, respectively. Using IMpRH panel, MMP19 was mapped to SSC5p12-q11 (closely linked to microsatellite DK) and MMP23B was mapped to SSC8q11-q12 (linked to microsatellite Sw2521). Quantitative real-time PCR showed that MMP19 was abundantly expressed in the liver, while MMP23B was strongly expressed in the ovarian and heart. Furthermore, both genes were all expressed increasingly in prenatal skeletal muscle during development. Three SNPs were detected by sequencing and PCR-RFLP methods, and association analysis indicated that C203T at exon 5 of MMP19 has a significant association with the blood parameters WBC (G/L) and IgG2 (mg/mL) (P<0.05), SNP C131T at exon 3 of MMP23B is significantly associated with the blood parameters HGB (g/L) and MCH (P<0.05), and A150G in exon 4 has no significant association with the economic traits in pigs.     

Crystal structures of BchU, a methyltransferase involved in bacteriochlorophyll c biosynthesis, and its complex with S-adenosylhomocysteine: implications for reaction mechanism

BchU plays a role in bacteriochlorophyll c biosynthesis by catalyzing methylation at the C-20 position of cyclic tetrapyrrole chlorin using S-adenosylmethionine (SAM) as a methyl source. This methylation causes red-shifts of the electronic absorption spectrum of the light-harvesting pigment, allowing green photosynthetic bacteria to adapt to low-light environments. We have determined the crystal structures of BchU and its complex with S-adenosylhomocysteine (SAH). BchU forms a dimer and each subunit consists of two domains, an N-terminal domain and a C-terminal domain. Dimerization occurs through interactions between the N-terminal domains and the residues responsible for the catalytic reaction are in the C-terminal domain. The binding site of SAH is located in a large cavity between the two domains, where SAH is specifically recognized by many hydrogen bonds and a salt-bridge. The electron density map of BchU in complex with an analog of bacteriochlorophyll c located its central metal near the SAH-binding site, but the tetrapyrrole ring was invisible, suggesting that binding of the ring to BchU is loose and/or occupancy of the ring is low. It is likely that His290 acts as a ligand for the central metal of the substrate. The orientation of the substrate was predicted by simulation, and allows us to propose a mechanism for the BchU directed methylation: the strictly conserved Tyr246 residue acts catalytically in the direct transfer of the methyl group from SAM to the substrate through an S(N)2-like mechanism.     

A comparison of the folding of two knotted proteins: YbeA and YibK

The extraordinary topology of proteins belonging to the alpha/beta-knot superfamily of proteins is unexpected, due to the apparent complexities involved in the formation of a deep trefoil knot in a polypeptide backbone. Despite this, an increasing number of knotted structures are being identified; how such proteins fold remains a mystery. Studies on the dimeric protein YibK from Haemophilus influenzae have led to the characterisation of its folding pathway in some detail. To complement research into the folding of YibK, and to address whether folding pathways are conserved for members of the alpha/beta-knot superfamily, the structurally similar knotted protein YbeA from Escherichia coli has been studied. A comprehensive thermodynamic and kinetic analysis of the folding of YbeA is presented here, and compared to that of YibK. Both fold via an intermediate state populated under equilibrium conditions that is monomeric and considerably structured. The unfolding/refolding kinetics of YbeA are simpler than those found for YibK and involve two phases attributed to the formation of a monomeric intermediate state and a dimerisation step. In contrast to YibK, a change in the rate-determining step on the unfolding pathway for YbeA is observed with a changing concentration of urea. Despite this difference, both proteins fold by a mechanism involving at least one sequential monomeric intermediate that has properties similar to that observed during the equilibrium unfolding. The rate of dimerisation observed for YbeA and YibK is very similar, as is the rate constant for formation of the kinetic monomeric intermediate that precedes dimerisation. The findings suggest that relatively slow folding and dimerisation may be common attributes of knotted proteins.     

Distinct determinants of tRNA recognition by the TrmD and Trm5 methyl transferases

TrmD and Trm5 are, respectively, the bacterial and eukarya/archaea methyl transferases that catalyze transfer of the methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 in tRNA to synthesize m1G37-tRNA. The m1G37 modification prevents tRNA frameshifts on the ribosome by assuring correct codon-anticodon pairings, and thus is essential for the fidelity of protein synthesis. Although TrmD and Trm5 are derived from unrelated AdoMet families and recognize the cofactor using distinct motifs, the question of whether they select G37 on tRNA by the same, or different, mechanism has not been answered. Here we address this question by kinetic analysis of tRNA truncation mutants that lack domains typically present in the canonical L shaped structure, and by evaluation of the site of modification on tRNA variants with an expanded or contracted anticodon loop. With both experimental approaches, we show that TrmD and Trm5 exhibit separate and distinct mode of tRNA recognition, suggesting that they evolved by independent and non-overlapping pathways from their unrelated AdoMet families. Our results also shed new light onto the significance of the m1G37 modification in the controversial quadruplet-pairing model of tRNA frameshift suppressors.     

Interaction of the tylosin-resistance methyltransferase RlmA II at its rRNA target differs from the orthologue RlmA I

RlmA^II methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-producer Streptomyces fradiae and the pathogen Streptococcus pneumoniae. Recombinant S. pneumoniae RlmA^II was purified and shown to retain its activity and specificity in vitro when tested on unmethylated 23 S rRNA substrates. RlmA^II makes multiple footprint contacts with nucleotides in stem-loops 33, 34 and 35, and does not interact elsewhere in the rRNA. Binding of RlmA^II to the rRNA is dependent on the cofactor S-adenosylmethionine (or S-adenosylhomocysteine). RlmA^II interacts with the same rRNA region as the orthologous enzyme RlmA^I that methylates at nucleotide G745. Differences in nucleotide contacts within hairpin 35 indicate how the two methyltransferases recognize their distinct targets.     

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

The RsmG methyltransferase is responsible for N⁷ methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.     

Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Bacterial TrmD and eukaryotic-archaeal Trm5 form a pair of analogous tRNA methyltransferase that catalyze methyl transfer from S-adenosyl methionine (AdoMet) to N(1) of G37, using catalytic motifs that share no sequence or structural homology. Here we show that natural and synthetic analogs of AdoMet are unable to distinguish TrmD from Trm5. Instead, fragments of AdoMet, adenosine and methionine, are selectively inhibitory of TrmD rather than Trm5. Detailed structural information of the two enzymes in complex with adenosine reveals how Trm5 escapes targeting by adopting an altered structure, whereas TrmD is trapped by targeting due to its rigid structure that stably accommodates the fragment. Free energy analysis exposes energetic disparities between the two enzymes in how they approach the binding of AdoMet versus fragments and provides insights into the design of inhibitors selective for TrmD.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

Crystal structure of the plant epigenetic protein arginine methyltransferase 10

Protein arginine methyltransferase 10 (PRMT10) is a type I arginine methyltransferase that is essential for regulating flowering time in Arabidopsis thaliana. We present a 2.6 Å resolution crystal structure of A. thaliana PRMT 10 (AtPRMT10) in complex with a reaction product, S-adenosylhomocysteine. The structure reveals a dimerization arm that is 12-20 residues longer than PRMT structures elucidated previously; as a result, the essential AtPRMT10 dimer exhibits a large central cavity and a distinctly accessible active site. We employ molecular dynamics to examine how dimerization facilitates AtPRMT10 motions necessary for activity, and we show that these motions are conserved in other PRMT enzymes. Finally, functional data reveal that the 10 N-terminal residues of AtPRMT10 influence substrate specificity, and that enzyme activity is dependent on substrate protein sequences distal from the methylation site. Taken together, these data provide insights into the molecular mechanism of AtPRMT10, as well as other members of the PRMT family of enzymes. They highlight differences between AtPRMT10 and other PRMTs but also indicate that motions are a conserved element of PRMT function.