SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy.

HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.

High Expression of Serine Hydroxymethyltransferase 2 Indicates Poor Prognosis of Gastric Cancer Patients.

BACKGROUND Serine hydroxymethyltransferase (SHMT) is the enzyme that catalyzes the reversible conversion of serine to glycine and tetrahydrofolate-bound one-carbon unit. Upregulation of SHMT2 has been observed in a variety of cancers, but the expression profile and clinical value of SHMT2 in gastric cancer (GC) are still unknown. MATERIAL AND METHODS In this study, SHMT2 expression was assessed in 130 patients with GC by immunohistochemistry (IHC). mRNA of SHMT2 in GC tissues and normal gastric epithelium was compared with qRT-PCR results. The correlations between SHMT2 and the clinicopathologic factors were analyzed with the chi-square test. Univariate analysis with Kaplan-Meier method was used to estimate the correlations between survival rate and clinicopathologic factors, including SHMT2. The independent prognostic biomarkers were confirmed by multivariate analysis using the Cox-regression hazard model. The function of SHMT2 in progression of GC was assessed by in vitro experiments. RESULTS The percentages of low and high expression of SHMT2 were 46.92% and 53.08%, respectively. SHMT2 mRNA in GC tissue was significantly higher than mRNA in the patient-paired adjacent tissues. In the clinical analysis, SHMT2 expression was notably associated with positive lymphatic invasion. High SHMT2 was also demonstrated to independently predict poor prognosis of GC. After silencing SHMT2, we proved that SHMT2 can promote proliferation and invasion of GC cells. CONCLUSIONS High SHMT2 promoted progression and was an independent prognostic biomarker of GC, suggesting that SHMT2 detection would be helpful for stratification of high-risk patients and thus directing personalized treatment.

Increased Expression of Serine Hydroxymethyltransferase 2 (SHMT2) is a Negative Prognostic Marker in Patients with Hepatocellular Carcinoma and is Associated with Proliferation of HepG2 Cells.

BACKGROUND Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon cell metabolism, including in liver cancer. However, the associations between SHMT2 expression at the gene and protein level and prognosis in patients with hepatocellular carcinoma (HCC) remains unknown. This study aimed to investigate the expression levels of SHMT2 in tumor tissue samples from patients with HCC and clinical outcome and the effects of silencing the expression of the SHMT2 gene in HepG2 cells. MATERIAL AND METHODS Expression levels of SHMT2 were evaluated in 144 cases of HCC using immunohistochemistry and correlated with clinicopathological factors using the chi-squared (χ²) test. The prognostic significance of SHMT2 expression was analyzed by univariate analysis and multivariate analysis. Twenty pairs of HCC tissue and adjacent normal liver tissue were compared for SHMT2 expression levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). HepG2 cells underwent SHMT2 gene silencing and MTT and transwell assays investigated cell proliferation and migration. Western blot was used to detect the expression of markers of epithelial-mesenchymal transition (EMT). RESULTS Expression levels of SHMT2 in HCC tissues were significantly correlated with tumor grade and hepatitis B virus (HBV) infection, and increased expression was an independent negative prognostic factor in patients with HCC (P=0.003). Increased expression of the SHMT2 gene promoted the proliferation and migration of the HepG2 HCC cell line. CONCLUSIONS Increased expression of SHMT2 was a negative prognostic biomarker in patients with HCC. Expression of the SHMT2 gene promoted the proliferation and migration of HepG2 HCC cells.

Role of Cytosolic Serine Hydroxymethyl Transferase 1 (SHMT1) in Phosphate-Induced Vascular Smooth Muscle Cell Calcification.

BACKGROUND/AIMS: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. METHODS: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. RESULTS: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. CONCLUSION: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA.

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.

[Analysis of mouse strain-dependent prepulse inhibition points to a role for Shmt1 (SHMT1) in mice and in schizophrenia].

Deficits in prepulse inhibition (PPI) are thought to be a biological trait of mental illnesses, including schizophrenia. It is known that the N-methyl-D-aspartate type glutamate (NMDA) receptor function affects PPI integrity and D-serine and glycine are typical endogenous co-agonists for the receptor. In parallel, we re-visited our prior quantitative trait loci (QTL) analysis study that examined C57BL/6 (B6) mice with high PPI and C3H/He (C3) with low PPI, and noticed that the genes encoding enzymes responsible for the productions of D-serine (serine racemase: Srr) and glycine (serine hydroxymethyltransferase 1: Shmt1) map to the chromosome 11 QTL. Therefore, we set out to examine whether brain interstitial fluid (ISF) levels of the two amino acids are different between the two mouse strains, using in vivo microdialysis. Recovery of D-serine and glycine from the dialysate of the frontal cortex was higher in B6 mice, which performed better in PPI, compared to C3 mice. Next, we analyzed the two genes, Srr and Shmt1. We then identified promoter polymorphisms in Shmt1 which elicit lower transcriptional activity in B6 compared to C3 mice. Human studies revealed higher expression levels of SHMT1 in the frontal cortex of postmortem brains from schizophrenics compared to controls, but no changes in SRR levels. In addition, genetic analysis detected a nominal association between SHMT1 and schizophrenia. These results suggest that Shmt1 (SHMT1) is one of the genetic components regulating PPI in mice and is relevant to schizophrenia susceptibility in humans.

Cardiac autonomic dysfunction: effects from particulate air pollution and protection by dietary methyl nutrients and metabolic polymorphisms.

BACKGROUND: Particulate air pollution is associated with cardiovascular mortality and morbidity. To help identify mechanisms of action and protective/susceptibility factors, we evaluated whether the effect of particulate matter <2.5 mum in aerodynamic diameter (PM(2.5)) on heart rate variability was modified by dietary intakes of methyl nutrients (folate, vitamins B(6) and B(12), methionine) and related gene polymorphisms (C677T methylenetetrahydrofolate reductase [MTHFR] and C1420T cytoplasmic serine hydroxymethyltransferase [cSHMT]). METHODS AND RESULTS: Heart rate variability and dietary data were obtained between 2000 and 2005 from 549 elderly men from the Normative Aging Study. In carriers of [CT/TT] MTHFR genotypes, the SD of normal-to-normal intervals was 17.1% (95% CI, 6.5 to 26.4; P=0.002) lower than in CC MTHFR subjects. In the same [CT/TT] MTHFR subjects, each 10-mug/m(3) increase in PM(2.5) in the 48 hours before the examination was associated with a further 8.8% (95% CI, 0.2 to 16.7; P=0.047) decrease in the SDNN. In [CC] cSHMT carriers, PM(2.5) was associated with an 11.8% (95% CI, 1.8 to 20.8; P=0.02) decrease in SDNN. No PM(2.5)-SSDN association was found in subjects with either [CC] MTHFR or [CT/TT] cSHMT genotypes. The negative effects of PM(2.5) were abrogated in subjects with higher intakes (above median levels) of B(6), B(12), or methionine. PM(2.5) was negatively associated with heart rate variability in subjects with lower intakes, but no PM(2.5) effect was found in the higher intake groups. CONCLUSIONS: Genetic and nutritional variations in the methionine cycle affect heart rate variability either independently or by modifying the effects of PM(2.5).

Serine hydroxymethyl transferase 1 stimulates pro-oncogenic cytokine expression through sialic acid to promote ovarian cancer tumor growth and progression.

High-grade serous (HGS) ovarian cancer accounts for 90% of all ovarian cancer-related deaths. However, factors that drive HGS ovarian cancer tumor growth have not been fully elucidated. In particular, comprehensive analysis of the metabolic requirements of ovarian cancer tumor growth has not been performed. By analyzing The Cancer Genome Atlas mRNA expression data for HGS ovarian cancer patient samples, we observed that six enzymes of the folic acid metabolic pathway were overexpressed in HGS ovarian cancer samples compared with normal ovary samples. Systematic knockdown of all six genes using short hairpin RNAs (shRNAs) and follow-up functional studies demonstrated that serine hydroxymethyl transferase 1 (SHMT1) was necessary for ovarian cancer tumor growth and cell migration in culture and tumor formation in mice. SHMT1 promoter analysis identified transcription factor Wilms tumor 1 (WT1) binding sites, and WT1 knockdown resulted in reduced SHMT1 transcription in ovarian cancer cells. Unbiased large-scale metabolomic analysis and transcriptome-wide mRNA expression profiling identified reduced levels of several metabolites of the amino sugar and nucleotide sugar metabolic pathways, including sialic acid N-acetylneuraminic acid (Neu5Ac), and downregulation of pro-oncogenic cytokines interleukin-6 and 8 (IL-6 and IL-8) as unexpected outcomes of SHMT1 loss. Overexpression of either IL-6 or IL-8 partially rescued SHMT1 loss-induced tumor growth inhibition and migration. Supplementation of culture medium with Neu5Ac stimulated expression of IL-6 and IL-8 and rescued the tumor growth and migratory phenotypes of ovarian cancer cells expressing SHMT1 shRNAs. In agreement with the ovarian tumor-promoting role of Neu5Ac, treatment with Neu5Ac-targeting glycomimetic P-3Fax-Neu5Ac blocked ovarian cancer growth and migration. Collectively, these results demonstrate that SHMT1 controls the expression of pro-oncogenic inflammatory cytokines by regulating sialic acid Neu5Ac to promote ovarian cancer tumor growth and migration. Thus, targeting of SHMT1 and Neu5Ac represents a precision therapy opportunity for effective HGS ovarian cancer treatment.

Structure-function relationship in serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine. The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate. The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

Molecular organization, catalytic mechanism and function of serine hydroxymethyltransferase--a potential target for cancer chemotherapy.

Serine hydroxymethyltransferase, a pyridoxal-5'-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-H4-folate. The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism. Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs. However, development of resistance to drugs, due to a variety of reasons, has necessitated the identification of alternate targets for cancer chemotherapy and serine hydroxymethyltransferase is one such potential target. A detailed study of the kinetics of interaction of serine and folate analogs with this enzyme revealed several unique features that can be exploited for the design of new chemotherapeutic agents. The pathways for the reversible unfolding of the dimeric Escherichia coli and the tetrameric sheep liver enzyme, although different, revealed a requirement for the cofactor in the final step for generating an active enzyme. The gly A gene of Escherichia coli has been shown to code for this enzyme. Analysis of available gene sequences indicate that serine hydroxymethyltransferase is one of the most highly conserved proteins. The isolation of the cDNA clones for the enzyme and their overexpression in heterologous systems has enabled the probing of the molecular mechanisms of catalysis and the role of lysine, arginine and histidine in cofactor, substrate(s) binding and in maintaining the structure of the protein. Recently, the three-dimensional structure of the human liver serine hydroxymethyltransferase has been published. This, along with the information already available, provides a framework for the rational design of drugs targeted specifically towards this enzyme.

Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39.

L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).

Psychosis and vulnerability to ECT-induced seizures.

Medical records of patients with major depressive disorders who had received electroconvulsive therapy (ECT) for the first time were studied to test the hypothesis that psychotic patients are more vulnerable to seizures than nonpsychotic patients. This hypothesis was based on studies suggesting a putative purinergic deficiency in psychosis. Results showed that the duration of ECT-induced seizures as a measure of seizure vulnerability was significantly longer in psychotic than in nonpsychotic depressive patients. The association applied for the first ECT as well as for the course of eight ECTs. These findings were still present when covariates such as age, electrical energy applied, dosage of methohexital and succinylcholine, and psychotropic medications such as neuroleptics, benzodiazepines, and tricyclics were included in the statistical analysis. The results are discussed in the context of the role of neurotransmitters such as glutamate, gamma-aminobutyric acid, adenosine, and dopamine on seizure vulnerability and psychosis.

The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1.

Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C2 cycle, namely, the conversion of two molecules of glycine into one molecule each of CO2, NH4+, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO2, but is virtually unaffected at elevated CO2. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G --> A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F1 progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.

Arabidopsis SHMT1, a serine hydroxymethyltransferase that functions in the photorespiratory pathway influences resistance to biotic and abiotic stress.

We found that a recessive mutation, shmt1-1, causes aberrant regulation of cell death resulting in chlorotic and necrotic lesion formation under a variety of environmental conditions. Salicylic acid-inducible genes and genes involved in H(2)O(2) detoxification were expressed constitutively in shmt1-1 plants in direct correlation with the severity of the lesions. The shmt1-1 mutants were more susceptible than control plants to infection with biotrophic and necrotrophic pathogens, developing severe infection symptoms in a high percentage of infected leaves. In addition, mutants carrying shmt1-1 or a loss-of-function shmt1-2 allele, were smaller and showed a greater loss of chlorophyll and greater accumulation of H(2)O(2) than wild-type plants when subjected to salt stress. SHMT1 was map-based cloned and found to encode a serine hydroxymetyltransferase (SHMT1) involved in the photorespiratory pathway. Our results indicate that this enzymatic activity plays a critical role in controlling the cell damage provoked by abiotic stresses such as high light and salt and in restricting pathogen-induced cell death, supporting the notion that photorespiration forms part of the dissipatory mechanisms of plants to minimize production of reactive oxygen species (ROS) at the chloroplast and to mitigate oxidative damage. Moreover, results shown here indicate that whereas production of ROS is an essential component of the hypersensitive defense response, the excessive accumulation of these toxic compounds impairs cell death containment and counteracts the effectiveness of the plant defenses to restrict pathogen infection.

Influence of pyruvate, threonine and phosphoethanolamine on activities of some acetaldehyde-producing enzymes.

Threonine (50 mg/100 g, i.p.) leads to increased hepatic threonine aldolase activity in rats, although endogenous ethanol concentrations remain stable. After pyruvate administration (50 mg/100 g, i.p.), endogenous blood ethanol levels are raised within 30 min, but return to normal at 60 min. The activity of threonine aldolase is decreased in the liver, whereas phosphoethanolamine lyase and pyruvate dehydrogenase activities remain unchanged. Phosphoethanolamine administration (23 mg/100 g, i.p.) did not change the endogenous ethanol concentration or pyruvate dehydrogenase, threonine aldolase and phosphoethanolamine lyase activities. Pyruvate appears to be a better precursor of acetaldehyde than threonine or phosphoethanolamine.

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.

Genetic interaction between a chaperone of small nucleolar ribonucleoprotein particles and cytosolic serine hydroxymethyltransferase.

Srp40p is a nonessential yeast nucleolar protein proposed to function as a chaperone for over 100 small nucleolar ribonucleoprotein particles that are required for rRNA maturation. To verify and expand on its function, genetic screens were performed for the identification of genes that were lethal when mutated in a SRP40 null background (srp40Delta). Unexpectedly, mutation of both cytosolic serine hydroxymethyltransferase (SHM2) and one-carbon tetrahydrofolate synthase (ADE3) was required to achieve synthetic lethality with srp40Delta. Shm2p and Ade3p are cytoplasmic enzymes producing 5,10-methylene tetrahydrofolate in convergent pathways as the primary source for cellular one-carbon groups. Nonetheless, point mutants of Shm2p that were catalytically inactive (i.e. failed to rescue the methionine auxotrophy of a shm2Delta ade3 strain) complemented the synthetic lethal phenotype, thus revealing a novel metabolism-independent function of Shm2p. The same Shm2p mutants exacerbated a giant cell phenotype observed in the shm2Delta ade3 strain suggesting a catalysis-independent role for Shm2p in cell size control, possibly through regulation of ribosome biogenesis via SRP40. Additionally, we show that the Sm-like protein Lsm5p, which as part of Lsm complexes participates in cytosolic and nuclear RNA processing and degradation pathways, is a multicopy suppressor of the synthetic lethality and of the specific depletion of box H/ACA small nucleolar RNAs from the srp40Delta shm2 ade3 strain. Finally, rat Nopp140 restored growth and stability of box H/ACA snoRNAs after genetic depletion of SRP40 in the synthetic lethal strain indicating that it is indeed the functional homolog of yeast Srp40p.

Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis.

Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals. Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp-like genes coding for homologues of the Escherichia coli leucine-responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT). SHMT catalyses interconversion of serine and glycine while transferring the resulting one-carbon unit into the C1 pool through methylene tetrahydrofolate. Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S-adenosyl-methionine (SAM). The results of experiments using L-ethionine suggest that SAM is involved in post-synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB. Truncated LrpA and LrpB peptides that have retained the DNA-binding domain but have lost the C-terminal half of the protein appear to act as repressors of glyA transcription and KinB-dependent sporulation. However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB. Our results indicate that lrpA-lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.