FDXR regulates TP73 tumor suppressor via IRP2 to modulate aging and tumor suppression.

Ferredoxin reductase (FDXR) is a mitochondrial flavoprotein that initiates electron transport from NADPH to several cytochromes P450 via two electron carriers, ferredoxin 1 (FDX1) and FDX2. FDXR is the sole ferredoxin reductase in humans and plays a critical role in steroidogenesis and biosynthesis of heme and iron-sulfur clusters. However, much less is known about the role of FDXR in cancer. Here, we show that FDXR plays a role in tumorigenesis by modulating expression of the tumor suppressor p73. By using genetically modified mouse models, we recently showed that mice deficient in either Fdxr or Trp73 had a shorter lifespan and were prone to spontaneous tumors as compared with wild-type (WT) mice. Interestingly, compound Trp73 +/- ;Fdxr +/- mice lived longer and developed fewer tumors when compared with Fdxr +/- or Trp73 +/- mice. Moreover, we found that cellular senescence was increased in Trp73 +/- and Fdxr +/- mouse embryonic fibroblasts (MEFs), which was further increased in Trp73 +/- ;Fdxr +/- MEFs, as compared with that in WT MEFs. As FDXR is regulated by p73, we examined whether there was a feedback regulation between p73 and FDXR. Indeed, we found that Trp73 expression was decreased by loss of Fdxr in MEFs and that FDXR is required for p73 expression in multiple human cancer cell lines independent of p53. Mechanistically, we found that loss of FDXR, via FDX2, increased expression of iron-binding protein 2 (IRP2), which subsequently repressed TP73 mRNA stability. We also showed that TP73 transcript contained an iron response element in its 3'UTR, which was required for IRP2 to destabilize TP73 mRNA. Together, these data reveal a novel regulation of p73 by FDXR via IRP2 and that the FDXR-p73 axis plays a critical role in aging and tumor suppression. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Methionine synthase reductase deficiency (CblE): A report of two patients and a novel mutation.

IMPORTANCE: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment. CLINICAL PRESENTATION: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them. INTERVENTION: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677-1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency. CONCLUSION: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme.

Mouse model for deficiency of methionine synthase reductase exhibits short-term memory impairment and disturbances in brain choline metabolism.

Hyperhomocysteinaemia can contribute to cognitive impairment and brain atrophy. MTRR (methionine synthase reductase) activates methionine synthase, which catalyses homocysteine remethylation to methionine. Severe MTRR deficiency results in homocystinuria with cognitive and motor impairments. An MTRR polymorphism may influence homocysteine levels and reproductive outcomes. The goal of the present study was to determine whether mild hyperhomocysteinaemia affects neurological function in a mouse model with Mtrr deficiency. Mtrr+/+, Mtrr+/gt and Mtrrgt/gt mice (3 months old) were assessed for short-term memory, brain volumes and hippocampal morphology. We also measured DNA methylation, apoptosis, neurogenesis, choline metabolites and expression of ChAT (choline acetyltransferase) and AChE (acetylcholinesterase) in the hippocampus. Mtrrgt/gt mice exhibited short-term memory impairment on two tasks. They had global DNA hypomethylation and decreased choline, betaine and acetylcholine levels. Expression of ChAT and AChE was increased and decreased respectively. At 3 weeks of age, they showed increased neurogenesis. In the cerebellum, mutant mice had DNA hypomethylation, decreased choline and increased expression of ChAT. Our work demonstrates that mild hyperhomocysteinaemia is associated with memory impairment. We propose a mechanism whereby a deficiency in methionine synthesis leads to hypomethylation and compensatory disturbances in choline metabolism in the hippocampus. This disturbance affects the levels of acetylcholine, a critical neurotransmitter in learning and memory.

Isolated remethylation disorders: do our treatments benefit patients?

Deficiency of 5,10-methylenetetrahydrofolate reductase (MTHFR), the very rare methionine synthase reductase (CblE) and methionine synthase (CblG) defects, and the recently identified CblD-variant-1 defect are primary remethylation defects characterized by an isolated defect in methionine synthesis without methylmalonic aciduria. The clinical signs are mainly neurological, and hematological signs are seen in CblE, CblG, and CblD-variant-1 defects. Patients with neonatal or early-onset disease exhibit acute neurological distress. Infants and children have unspecific mental retardation, often with acquired microcephaly. Without appropriate therapy, they may experience acute or rapidly progressive neurological deterioration, which may be fatal. Adolescents and adults show normal development or mild developmental delay initially and then experience rapid neurological or behavioral deterioration. A few patients may have signs of subacute combined degeneration of the spinal cord. Adults may be asymptomatic or present with isolated thromboembolism. All patients with suspected remethylation disorders should receive emergency treatment with parenteral administration of hydroxocobalamin and folate supplements combined with betaine orally. The long-term treatment of CblE, CblG, and CblD-variant-1 defects consists of parenterally administered hydroxocobalamin and orally administered folate and betaine supplements, whereas patients with MTHFR deficiency require long-term oral folate and betaine supplements. Long-term oral methionine therapy should also be considered. Early treatment may lead to a favorable outcome with developmental recovery and prevention of further neurological deterioration. In contrast, most late-treated patients have severe and irreversible neuromotor impairments. Hematological abnormalities are easily corrected.

[A rare inborn error of intracellular processing of cobalamine presenting with microcephalus and megaloblastic anemia: a report of 3 children]. / Mikrozephalus und megaloblastäre Anämie als Leitsymptome einer seltenen angeborenen Störung des intrazellulären Kobalamin-Processings--3 Kasuistiken.

INTRODUCTION: Defects of methionine synthase or methionine synthase reductase result in an impaired remethylation of homocysteine to methionine. Patients present with megaloblastic anemia, failure to thrive and various neurological manifestations including mental retardation, cerebral atrophy, muscular hypotonia or hypertonia, ataxia, seizures, nystagmus and visual disturbances. PATIENTS: We report on three children (two girls, one boy), aged 3.5-7.5 years, who presented with severe megaloblastic anemia, micro-cephalus and partly nystagmus (2/3) due to a rare inborn error of remethylation. RESULTS: Methionine synthase reductase deficiency, cblE type of homocystinuria (OMIM 236270), is a rare autosomal recessive inherited disorder described only in 14 patients worldwide. Metabolic hallmarks of the disease are hyperhomocysteinemia (median 98 micromol/l, normal range <15) without methylmalonic aciduria but often hypomethioninemia. The patients described here were diagnosed at ages of 2-18 months. The importance of an early recognition of this possibly underdiagnosed congenital disease is stressed. Treatment consisted of the application of hydroxocobalamine (1-2 mg weekly, i.m.), betaine (100-200 mg/kg daily, p.o.), folate (5-10 mg daily, p.o.) and intensive physical therapy. CONCLUSION: Defects of intracellular processing of cobalamine must be considered in all patients with neurological symptoms in combination with megaloblastic anemia. Measurements of homocysteine and methionine in plasma as well as methylmalonic acid in urine is required for confirming the diagnosis. Early treatment im-proves the outcome, although mental disability may not be prevented. Treatment has a positive impact on megaloblastic anemia but only slight effect on hyperhomocysteinemia. The long-term cardiovascular risk of hyperhomocysteinemia in cblE deficient patients is not known yet.

cblE type of homocystinuria due to methionine synthase reductase deficiency: functional correction by minigene expression.

The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.

Small changes in the activity of chloroplastic NADP(+)-dependent ferredoxin oxidoreductase lead to impaired plant growth and restrict photosynthetic activity of transgenic tobacco plants.

A ferredoxin-NADP+ oxidoreductase (FNR) cDNA from tobacco (Nicotiana tabacum cv. Samsun) was cloned and sequenced. Comparison of the deduced amino acid sequence revealed high identity to FNR proteins from Capsicum annuum, Pisum sativum, Spinacia oleracea and Vicia faba. Transgenic tobacco plants were generated that constitutively express the FNR cDNA in reverse orientation between the CaMV 35S promoter and the polyadenylation signal of the octopine synthase gene. Plants expressing the FNR antisense gene showed lower levels of FNR mRNA and protein accumulation, which was paralleled by a decrease in FNR activity. As a consequence, NADPH levels declined whereas NADP+ levels increased, leading to an unaltered NADP(H) pool. Growth rates, chlorophyll content and net CO2 uptake rates at high and low irradiances were strongly reduced in FNR antisense tobacco plants. These changes were accompanied by an over-reduced state of P700 as estimated by absorption changes at 820 nm. FNR control coefficients determined for the photosynthetic rate at saturating (C(R) = 0.94) and limiting (C(R) = 0.70) light conditions revealed a prominent role of this reductase in the regulation of photosynthesis.

CblE type of homocystinuria due to methionine synthase reductase deficiency: clinical and molecular studies and prenatal diagnosis in two families.

The cblE type of homocystinuria is a rare autosomal recessive disorder, which manifests with megaloblastic anaemia and developmental delay in early childhood. This disease is caused by a defect in reductive activation of methionine synthase (MTR). Our study was directed at clinical, biochemical, enzymatic and molecular characterization of two Czech patients with the cblE type of homocystinuria. Case 1 involves a 20-year-old mentally retarded patient who presented with megaloblastic anaemia at 10 weeks of age. She was treated with folates and vitamin B12, and subsequent attempts to cease administration of folates led to recurrence of megaloblastic anaemia. Biochemical features included severe hyperhomocysteinaemia and hypomethioninaemia and in fibroblasts defective formation of methionine from formate, and no complementation with cblE cells. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1459G>A (G487R) and a 2bp insertion (c.1623-1624insTA). Case 2 involves an 8-year-old girl with nystagmus and developmental delay in whom megaloblastic anaemia was detected at 11 weeks of age. Severe hyperhomocysteinaemia with normal methionine levels was found and enzymatic and complementation studies confirmed the cblE defect. This patient is homozygous for a 140 bp insertion (c.903-904ins140). The insertion is caused by a T>C transition within intron 6 of the MTRR gene, which presumably leads to activation of an exon splicing enhancer. In the families of both patients, enzymatic and mutation analyses were successfully used for prenatal diagnosis. Our study expands the knowledge of the phenotypic and genotypic variability of the cblE type of homocystinuria and supports the concept that this disorder is caused by mutations in the MTRR gene.

Molecular basis for methionine synthase reductase deficiency in patients belonging to the cblE complementation group of disorders in folate/cobalamin metabolism.

Methionine synthase reductase (MSR) deficiency is an autosomal recessive disorder of folate/cobalamin metabolism leading to hyperhomocysteinemia, hypo- methioninemia and megaloblastic anemia. Deficiency in MSR activity occurs as the result of a defect in the MSR enzyme, which is required for the reductive activation of methionine synthase (MS). MS itself is responsible for the folate/cobalamin-dependent conversion of homo- cysteine to methionine. We have recently cloned the cDNA corresponding to the MSR protein, a novel member of the ferredoxin-NADP(+)reductase (FNR) family of electron transferases. We have used RT-PCR, heteroduplex, single-strand conformation poly- morphism (SSCP) and DNA sequence analyses to reveal 11 mutations in eight patients from seven families belonging to the cblE complementation group of patients of cobalamin metabolism that is defective in the MSR protein. The mutations include splicing defects leading to large insertions or deletions, as well as a number of smaller deletions and point mutations. Apart from an intronic substitution found in two unrelated patients, the mutations appear singular among individuals. Of the eleven, three are nonsense mutations, allowing for the identification of two patients for whom little if any MSR protein should be produced. The remaining eight involve point mutations or in-frame disruptions of the coding sequence and are distributed throughout the coding region, including proposed FMN, FAD and NADPH binding sites. These data demonstrate a unique requirement for MSR in the reductive activation of MS.

Molecular genetics of homocysteine metabolism.

Recent genetic studies have led to the characterization of molecular determinants contributing to the pathogenesis of hyperhomocysteinemia. In this article we summarize the current insights into the molecular genetics of severe, moderate and mild hyperhomocysteinemia. We will consider deficiencies of the trans-sulfuration enzyme cystathionine beta-synthase (gene symbol: CBS), and the disturbances of the remethylation enzymes 5, 10-methylenetetrahydrofolate reductase (gene symbol: MTHFR), methionine synthase (gene symbol: MTR), and the recently identified methionine synthase reductase (gene symbol: MTRR). Furthermore, we will focus on clinically important genetic polymorphisms which are highly prevalent and thus of potential general interest.