Fumarylacetoacetate Hydrolase Research Articles

Correcting tyrosinaemia via a point mutation.

In an adult mouse model of tyrosinaemia, a base editor correcting an A-to-G splice-site mutation in the Fah gene restores the translation of the functional enzyme, promoting the repopulation of the liver with the corrected cells.

ExVivo/Invivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

SummaryAdeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.

Hereditary tyrosinemia type 1 in children

Hereditary metabolic disorders include a group of diseases (more than 400) when a defect of a particular gene changes the metabolic process leading either to the accumulation of unwanted metabolites, or to a deficiency of a substance. This group also includes hereditary tyrosinemia type 1, a severe defect of tyrosine metabolism caused by deficiency of fumarylacetoacetate hydrolase (FAH) – the last enzyme of tyrosine catabolic pathway. Tyrosinemia type 1 is an autosomal recessive disorder. This paper presents a review of literature on the current state of diagnosticis and approaches to treatment of tyrosinemia using nitisinone and a low-protein diet, as well as the analysis of clinical manifestations and laboratory diagnostics of hereditary tyrosinemia type 1 in 17 children.

In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice

The murine host has remained a readily available and ethically acceptable model for the study of human diseases and therapeutic testing. Immunodeficient mouse models support engraftment of human hematopoietic stem cells (HSC) but with limitation in efficiency and mature lineage representation. Combined knock-in of several non-crossreactive human cytokines (M-CSF, IL3/GM-CSF, and Thrombopoietin) into the corresponding murine loci in the SRG strain (in short termed "MISTRG") has enhanced engraftment and maintenance of human HSCs with multi-lineage differentiation (Rongvaux et al. Annu Rev Immunol 2013, Deng et al. Nature 2015, Saito et al. Blood 2016, Theocharides et al. Haematologica 2016). Despite robust HSC engraftment and myelo- and erythropoiesis in bone marrow (BM), all humanized immunodeficient mouse models lack of mature human red blood cells (RBC), platelets, and myeloid cells in peripheral blood (PB) (Rahmig et al. Stem Cell Reports 2016, Yurino et al. Stem Cell Reports 2016, Song et al. Nat Commun 2019). Yet, full maturation and representation of all myeloid lineages in PB is essential to study diseases of the HSC, such as MDS, and of the RBC, such as sickle cell anemia or malaria. With universal absence of a murine adaptive immune system the culprit is likely the murine host's innate immune system. Previous studies have shown that treatment of engrafted mice with liposomal clodronate that abrogates murine (and human) macrophages, with or without cobra venom factor that eliminates complement, can increase mature human circulating RBC, but only transiently and with significant toxicity. We first sought to determine the site of huRBC sequestration and destruction. Intravital imaging after injection of CFSE labelled huRBC identified the murine liver as the major site of RBC destruction. While muRBC rapidly circulate through the liver circulation, huRBC have greatly increased transit times and are sequestered in liver vessels. We hypothesized that humanization of the murine host's liver could potentially alleviate huRBC sequestration and significantly increase circulating huRBC. In previous studies deletion of fumarylacetoacetate hydrolase (Fah) in the Rag-/-Il2rg-/- background has allowed humanization of the liver and served to study diseases such as malaria (Vaughan et al. J Clin Invest 2012). The liver is the site of synthesis of numerous proteins, some of which directly impact hematopoiesis and blood cells, such as complement. We deleted the Fah gene via CRISPR/Cas9 in MISTRG mice and crossed MISTRG-Fah-/- mice to homozygosity (MISTRGFah). MISTRGFah are viable, fertile, and healthy when maintained on drinking water supplemented with 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC), that blocks tyrosine metabolism upstream of Fah and prevents buildup of hepatotoxic metabolites. At 8 weeks of age we implanted MISTRGFah mice with commercially available, adult human hepatocytes (HuHep) via direct injection into the splenic vein, followed by gradual withdrawal of NTBC water. Regulated buildup of intracellular fumarylacetoacetate results in death of murine Fah-/- hepatocytes and regeneration with HuHep with up to 90% repopulation by HuHep (Azuma et al. Nature biotechnology 2007). When plasma human albumin levels reached 2mg/dL, indicative of significant (~80%) HuHep repopulation, we sublethally (80cGy) irradiated HuHepMISTRGFah mice and engrafted each mouse with 105 fetal liver (FL) derived CD34+ cells. 10 weeks post transplantation, mice were analyzed for engraftment and specifically erythroid maturation in PB. HuHepMISTRGFah mice had significantly higher levels of BM and interestingly spleen erythropoiesis and circulating huRBC in PB (Fig.1 a). CD235a+ huRBC in HuHepMISTRGFah mice are enucleated (Hoechst neg) and mature as evident by loss of CD49d (ITGA4) and gain of Band3 staining (Hu et al. Blood 2013) (Fig.1 b). Interestingly, human erythroid cells in MISTRG but not HuHepMISTRGFah mice are coated with murine complement C3 (muC3) (Fig.1 c) suggesting that liver humanization results in loss of muC3 expression. In conclusion, we have generated the first humanized mouse model with fully mature, circulating huRBC when engrafted with human CD34+ stem and progenitor cells. Ongoing studies are testing the applicability of this model to MDS and sickle cell disease. Disclosures Flavell: SMOC: Equity Ownership; Zai labs: Consultancy; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; Rheos Biomedicines: Equity Ownership.

The autoimmune response elicited by mouse hepatitis virus (MHV-A59) infection is modulated by liver tryptophan-2,3-dioxygenase (TDO)

In a previous work we demonstrated that inhibition of mouse indoleamine 2,3-dioxygenase (IDO) by methyltryptophan (MT) exacerbated the pathological actions of mouse hepatitis virus (MHV-A59) infection, suggesting that tryptophan (TRP) catabolism was involved in viral effects. Since there is a second enzyme that dioxygenates TRP, tryptophan-2, 3-dioxygenase (TDO), which is mainly located in liver, we decided to study its role in our model of MHV-infection.Results showed that in vivo TDO inhibition by LM10, a derivative of 3-(2-(pyridyl) ethenyl) indole, resulted in a decrease of anti- MHV Ab titers induced by the virus infection. Besides, a reduction of some alarmin release, i.e, uric acid and high-mobility group box1 protein (HMGB1), was observed. Accordingly, since alarmin liberation was related to the expression of autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), these autoAb also diminished. Moreover, PCR results indicated that TDO inhibition did not abolish viral replication. Furthermore, histological liver examination did not reveal strong pathologies, whereas mouse survival was hundred percent in control as well as in MHV-infected mice treated with LM10.Data presented in this work indicate that in spite of the various TDO actions already described, specific TDO blockage could also restrain some MHV actions, mainly suppressing autoimmune reactions. Such results should prompt further experiments with various viruses to confirm the possible use of a TDO inhibitor such as LM-10 to treat either viral infections or even autoimmune diseases triggered by a viral infection.

Sequence, structure and function-based classification of the broadly conserved FAH superfamily reveals two distinct fumarylpyruvate hydrolase subfamilies.

Fumarylacetoacetate hydrolase (FAH) superfamily proteins are found ubiquitously in microbial pathways involved in the catabolism of aromatic substances. Although extensive bioinformatic data on these proteins have been acquired, confusion caused by problems with the annotation of these proteins hinders research into determining their physiological functions. Here we classify 606 FAH superfamily proteins using a maximum likelihood (ML) phylogenetic tree, comparative gene-neighbourhood patterns and in vitro enzyme assays. The FAH superfamily proteins used for the analyses are divided into five distinct subfamilies, and two of them, FPH-A and FPH-B, contain the majority of the proteins of undefined function. These subfamilies include clusters designated FPH-I and FPH-II, respectively, which include two distinct types of fumarylpyruvate hydrolase (FPH), an enzyme involved in the final step of the gentisate pathway. We determined the crystal structures of these FPH enzymes at 2.0 Å resolutions and investigate the substrate binding mode by which these types of enzymes can accommodate fumarylpyruvate as a substrate. Consequentially, we identify the molecular signatures of the two types of FPH enzymes among the broadly conserved FAH superfamily proteins. Our studies allowed us to predict the relationship of unknown FAH superfamily proteins using their sequence information.

Tissue-specific FAH deficiency alters sleep–wake patterns and results in chronic tyrosinemia in mice

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyrosine catabolism, and mutations in the FAH gene are associated with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. In a behavioral screen of N-ethyl-N-nitrosourea mutagenized mice we identified a mutant line which we named "swingshift" (swst, MGI:3611216) with a nonsynonymous point mutation (N68S) in Fah that caused age-dependent disruption of sleep-wake patterns. Mice homozygous for the mutation had an earlier onset of activity (several hours before lights off) and a reduction in total activity and body weight when compared with wild-type or heterozygous mice. Despite abnormal behavioral entrainment to light-dark cycles, there were no differences in the period or phase of the central clock in mutant mice, indicating a defect downstream of the suprachiasmatic nucleus. Interestingly, these behavioral phenotypes became milder as the mice grew older and were completely rescued by the administration of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. Mechanistically, the swst mutation had no effect on the enzymatic activity of FAH, but rather promoted the degradation of the mutant protein. This led to reduced FAH protein levels and enzymatic activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine-but not methionine, phenylalanine, or succinylacetone-increased in homozygous mice, suggesting that swst mutants provide a model of mild, chronic HT1.

TYROSINEMIA I TYPE (LITERATURE REVIEW) WITH A DESCRIPTION OF THE CLINICAL CASE

Modern ideas about the most severe variant of hereditary tyrosinemia are reflected -- its first type related to orphan diseases (the population frequency of occurrence is 1: 100,000-120,000 live newborns). The latest data on etiology is highlighted (mutations in the gene for the enzyme fumarylacetoacetase (FAA), leading to a deficiency of fumarylacetohydrolase (FAH), the last enzyme in tyrosine catabоlism; the genetic mutation is localized on human chromosome 15q23-q25 and consists of 14 exons) and the mechanisms of development of the disease ( the bypass abnormal pathway of tyrosine metabolism with the formation of highly toxic and carcinogenic metabolites -- succinyl acetoacetate (SAA) and succinylacetone (SA), others; substrate accumulation mainly in FAH-deficient hepatocytes and proximal renal tubule cells; violation of the processes of gluconeogenesis, ammonia detoxification and hepatic protein synthesis), which determine different clinical variants and symptom polymorphism. A modern classification of type I tyrosinemia and a detailed protocol for diagnostic examination of the patient, including instrumental methods, laboratory monitoring of highly toxic derivatives of the tyrosine metabolic pathway and molecular genetic studies, are presented. Denotes a range of diseases that should be carried out a differential diagnosis of tyrosinemia. The modern concept of treatment of this orphan disease (protein-free diet, specialized amino acid mixtures, hepatoprotectors, vitamin D and others) is laid out with an emphasis on enzyme replacement therapy with nitisinone, an enzyme inhibitor (4-hydroxyphenylpyruvate dioxygenase) of the first phase of tyrosine degradation. A description of a clinical case and a 15-year-old teenager with a hereditary tyrosinemia type I who received a low protein diet, the amino acid mixture «XPHEN TYR Tyrosidone» (Nutricia) and nitisinone (Orfadin®).

Genetic Analysis of Tyrosinemia Type 1 and Fructose-1, 6 Bisphosphatase Deficiency Affected in Pakistani Cohorts

Background: Inborn errors of metabolism are inherited disorders that present in early childhood and are usually caused by monogenic recessive mutations in specific enzymes that metabolize dietary components. Distinct mutations are present in specific populations.Objective: To determine which genomic variants are present in Pakistani cohorts with hepatorenal tyrosinemia type 1 (HT1) and fructose 1,6-bisphosphatase deficiency (FBPD).Materials and Methods: We sequenced the fumaryl acetoacetate hydrolase encoding gene (FAH) including flanking regions in four unrelated HT1 cohorts and the fructose 1,6-bisphosphatase gene (FBP1) in eight FBPD cohorts.Results: We mapped two recessive mutations in FAH gene for HT1; c.1062 + 5G > A(IVS12 + 5G > A) in three families and c.974C > T(pT325M) in one. We identified three mutations in FBP1 gene; c.841G > A(p.E281K) in five FBPD families, c.472C > T(p.R158W) in two families and c.778G > A(p.G260R) in one.Conclusion: Knowledge of common variants for HTI and FBDP in our study population can be used in the future to build a diagnostic algorithm.

Mutational spectrum of Mexican patients with tyrosinemia type 1: In silico modeling and predicted pathogenic effect of a novel missense FAH variant.

BackgroundTyrosinemia type 1 (HT1, MIM#276700) is caused by a deficiency in fumarylacetoacetate hydrolase (FAH) and it is associated with severe liver and renal disfunction. At present, the mutational FAH (15q25.1, MIM*613871) spectrum underlying HT1 in the Mexican population is unknown. The objective of this study was to determine the FAH genotypes in eight nonrelated Mexican patients with HT1, who were diagnosed clinically.MethodsSequencing of FAH and their exon–intron boundaries and in silico protein modeling based on the crystallographic structure of mouse FAH.ResultsWe identified pathogenic variants in 15/16 studied alleles (93.8%). Nine different variants were found. The most commonly detected HT1‐causing allele was NM_000137.2(FAH):c.3G > A or p.(?) [rs766882348] (25%, n = 4/16). We also identified a novel missense variant NM_000137.2(FAH):c.36C > A or p.(Phe12Leu) in a homozygous patient with an early and fatal acute form. The latter was classified as a likely pathogenic variant and in silico protein modeling showed that Phe‐12 residue substitution for Leu, produces a repulsion in all possible Leu rotamers, which in turn would lead to a destabilization of the protein structure and possible loss‐of‐function.ConclusionHT1 patients had a heterogeneous mutational and clinical spectrum and no genotype–phenotype correlation could be established.

Evolution of tyrosinemia type 1 disease in patients treated with nitisinone in Spain.

Treatment with nitisinone (NTBC) has brought about a drastic improvement in the treatment and prognosis of hereditary tyrosinemia type I (HT1). We conducted a retrospective observational multicentric study in Spanish HT1 patients treated with NTBC to assess clinical and biochemical long-term evolution.We evaluated 52 patients, 7 adults and 45 children, treated with NTBC considering: age at diagnosis, diagnosis by clinical symptoms, or by newborn screening (NBS); phenotype (acute/subacute/chronic), mutational analysis; symptoms at diagnosis and clinical course; biochemical markers; doses of NTBC; treatment adherence; anthropometric evolution; and neurocognitive outcome.The average follow-up period was 6.1 ± 4.9 and 10.6 ± 5.4 years in patients with early and late diagnosis respectively. All patients received NTBC from diagnosis with an average dose of 0.82 mg/kg/d. All NBS-patients (n = 8) were asymptomatic at diagnosis except 1 case with acute liver failure, and all remain free of liver and renal disease in follow-up. Liver and renal affectation was markedly more frequent at diagnosis in patients with late diagnosis (P < .001 and .03, respectively), with ulterior positive hepatic and renal course in 86.4% and 93.2% of no-NBS patients, although 1 patient with good metabolic control developed hepatocarcinoma.Despite a satisfactory global nutritional evolution, 46.1% of patients showed overweight/obesity. Interestingly lower body mass index was observed in patients with good dietary adherence (20.40 ± 4.43 vs 24.30 ± 6.10; P = .08) and those with good pharmacological adherence (21.19 ± 4.68 vs 28.58 ± 213.79).intellectual quotient was ≥85 in all NBS- and 68.75% of late diagnosis cases evaluated, 15% of which need pedagogical support, and 6.8% (3/44) showed school failure.Among the 12 variants identified in fumarylacetoacetate hydrolase gene, 1 of them novel (H63D), the most prevalent in Spanish population is c.554–1 G>T.After NTBC treatment a reduction in tyrosine and alpha-fetoprotein levels was observed in all the study groups, significant for alpha-fetoprotein in no NBS-group (P = .03), especially in subacute/chronic forms (P = .018).This series confirms that NTBC treatment had clearly improved the prognosis and quality of life of HT1 patients, but it also shows frequent cognitive dysfunctions and learning difficulties in medium-term follow-up, and, in a novel way, a high percentage of overweight/obesity.

Modulation of serotonin signaling by the putative oxaloacetate decarboxylase FAHD-1 in Caenorhabditis elegans.

Human fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) is a mitochondrial oxalocatate decarboxylase, the first of its kind identified in eukaryotes. The physiological role of FAHD1 in other eukaryotes is still poorly understood. In C. elegans loss of the FAHD1 ortholog FAHD-1 was reported to impair mitochondrial function, locomotion and egg-laying behavior, yet the underlying mechanisms remained unclear. Using tissue-specific rescue of fahd-1(-) worms, we find that these phenotypic abnormalities are at least in part due to fahd-1’s function in neurons. Moreover, we show that egg-laying defects in fahd-1(-) worms can be fully rescued by external dopamine administration and that depletion of fahd-1 expression induces expression of several enzymes involved in serotonin biosynthesis. Together, our results support a role for fahd-1 in modulating serotonin levels and suggest this protein as a novel link between metabolism and neurotransmitter signaling in the nervous system. Finally, we propose a model to explain how a metabolic defect could ultimately lead to marked changes in neuronal signaling.

Hereditary tyrosinemia type I–associated mutations in fumarylacetoacetate hydrolase reduce the enzyme stability and increase its aggregation rate

More than 100 mutations in the gene encoding fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1), a metabolic disorder characterized by elevated blood levels of tyrosine. Some of these mutations are known to decrease FAH catalytic activity, but the mechanisms of FAH mutation–induced pathogenicity remain poorly understood. Here, using diffusion ordered NMR spectroscopy, cryo-EM, and CD analyses, along with site-directed mutagenesis, enzymatic assays, and molecular dynamics simulations, we investigated the putative role of thermodynamic and kinetic stability in WT FAH and a representative set of 19 missense mutations identified in individuals with HT1. We found that at physiological temperatures and concentrations, WT FAH is in equilibrium between a catalytically active dimer and a monomeric species, with the latter being inactive and prone to oligomerization and aggregation. We also found that the majority of the deleterious mutations reduce the kinetic stability of the enzyme and always accelerate the FAH aggregation pathway. Depending mainly on the position of the amino acid in the structure, pathogenic mutations either reduced the dimer population or decreased the energy barrier that separates the monomer from the aggregate. The mechanistic insights reported here pave the way for the development of pharmacological chaperones that target FAH to tackle the severe disease HT1.

Hereditary tyrosinemia type I-associated mutations in fumarylacetoacetate hydrolase reduce the enzyme stability and increase its aggregation rate

Hereditary tyrosinemia type I-associated mutations in fumarylacetoacetate hydrolase reduce the enzyme stability and increase its aggregation rate

HBx-K130M/V131I Promotes Liver Cancer in Transgenic Mice via AKT/FOXO1 Signaling Pathway and Arachidonic Acid Metabolism.

Chronic hepatitis B viral (HBV) infection remains a high underlying cause for hepatocellular carcinoma (HCC) worldwide, while the genetic mechanisms behind this remain unclear. This study elucidated the mechanisms contributing to tumor development induced by the HBV X (HBx) gene of predominantly Asian genotype B HBV and its common HBx variants. To compare the potential tumorigenic effects of K130M/V131I (Mut) and wild-type (WT) HBx on HCC, the Sleeping Beauty (SB) transposon system was used to deliver HBx Mut and WT into the livers of fumarylacetoacetate hydrolase (Fah)-deficient mice and in the context of transformation related protein 53 (Trp53) deficiency. From our results, HBx Mut had a stronger tumorigenic effect than its WT variant. Also, inflammation, necrosis, and fibrosis were evident in HBx experimental animals. Reduction of forkhead box O1 (FOXO1) with increased phosphorylation of upstream serine/threonine kinase (AKT) was detected under HBx Mut overexpression. Thus, it is proposed that HBx Mut enhances disease progression by reducing FOXO1 via phosphorylation of AKT. At the metabolomic level, HBx altered the expression of genes that participated in arachidonic acid (AA) metabolism, as a result of inflammation via accumulation of proinflammatory factors such as prostaglandins and leukotriene in liver. Taken together, the increased rate of HCC observed in chronic hepatitis B patients with K130M/V131I-mutated X protein, may be due to changes in AA metabolism and AKT/FOXO1 signaling. IMPLICATIONS: Our findings suggested that HBx-K130M/V131I-mutant variant promoted HCC progression by activating AKT/FOXO1 pathway and inducing stronger inflammation in liver via AA metabolism.

Screening for hereditary tyrosinemia and genotype analysis in newborns

To analyze the results of screening for hereditary tyrosinemia (HT) in newborns and its clinical features and genotype. The HT screening was conducted among 2 188 784 newborns from November 2013 to November 2018. The tyrosine (TYR)/ succinylacetone (SA) levels were detected by tandem mass spectrometry (MS-MS). The clinical characteristics, genetic results and following up data of identified patients were analyzed. The normal ranges (0.5%-95.5%) of TYR and SA were 34.5-280.0 μmol/L and 0.16-2.58 μmol/L, respectively. Three HT cases were confirmed with a detection rate of 1∶729 595. There was 1 case of tyrosinemia type Ⅰ (HTⅠ) (homozygous variations of c.455G>A in FAH gene), 1 case of tyrosinemia type Ⅱ(HTⅡ) (heterozygous variations of c.890G>T and c.408+1G>A in TAT gene), and 1 case of tyrosinemia type Ⅲ (HT Ⅲ) (homozygous variations of c.257T>C in HPD gene). The variations of c.890G>T, c.4081G>A of TAT and c.257T>C of HPD were novel. The positive predictive value of the screening was 3.4%. Case 1 (HTⅠ) with TYR and SA values of 666.9 μmol/L and 3.87 μmol/L respectively, presented cholestasis, mild elevated of liver enzyme and lactic acid, who were although fed with TYR and phenylalanine free milk, but died at 2 months of age. Case 2 (HTⅡ) with higher TYR (625.6 μmol/L) and normal SA at screening, received medical milk treatment; during the 7 months of follow-up the baby showed normal score of Bayley assessment and normal TYR without eye and skin symptoms. Case 3 (HT Ⅲ) with TYR of 1035.3 μmol/L and normal SA at screening; during the 29 months of follow-up the value of TYR fluctuated from 532.1 μmol/L to 1060.3 μmol/L due to irregular medical milk treatment, while the score of Bayley assessment was normal. HT is rare in the southern Chinese population, and the gene spectrum is scattered. Early treatment with nitisinone is recommended in children with HTⅠ, otherwise the prognosis is poor; the prognosis of children with HTⅡ is good when early treated with special diet; the prognosis of children with HTⅢ needs to be determined with more data.

Senescence suppressed proliferation of host hepatocytes is precondition for liver repopulation

In the fumarylacetoacetate hydrolase deficient (Fah−/−) mouse, massive liver repopulation can be easily obtained after transplanted hepatocytes. Understanding the mechanisms of complete liver repopulation in Fah−/− mice will be useful for future clinical application. Here, we found that the endogenous hepatocytes in liver of Fah−/− mice undertook senescence during the time of tyrosinemia symptoms. Increase of senescent hepatocytes in Fah−/− mice provided proliferative advantage to the transplanted hepatocytes. Importantly, senescent hepatocytes upregulated the expression of extracellular matrix enzyme, contributing to degradation of extracellular matrix components and weakness of cell adhesion and connection. The liver exhibiting a loose architecture provided the space for the engraftment and expansion of transplanted hepatocytes. These findings underscore the underlying mechanisms of completed liver repopulation in Fah−/− mice. Senescence followed by loose hepatic parenchyma is a preconditioning for liver repopulation, which would be a promising strategy to achieve therapeutic liver repopulation in clinical settings.

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Study of a Nano-Oleuropein’s Effect on the TCA Cycle`s Protein Expression in the Breast Cancer Cell Line Using Proteomics

Breast cancer is the most common cancer and a common cause of death, which occurs due to cancer among women in the world. Cancer cells need a lot of energy to their uncontrolled growth, so it seems that the expression of the enzyme in the Krebs cycle is changing. There are some reports about mutations and altered expression of succinate dehydrogenase, fumarate Hydratase, and isocitrate dehydrogenase in human cancers. This research aimed to investigate the role of magnetite nanoparticle Oleuropein on the Krebs cycle proteins expression on the breast cancer cell line. Oleuropein is one of the polyphenolic components in olive trees and has some benefits in some diseases, including cancer. In addition to testing the viability test MTT (3- 4,5 Dimethylthiazol-2-yl -2,5-diphenyltetrazolium bromide) assay, in three levels of Oleuropein 0ppm, 300ppm, 600ppm proteomics analysis was also performed in cell line MCF7 in this study. The results of differential protein spots identification into two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS-MALDI-TOF-TOF), showed that fumarylacetoacetate hydrolase, succinate-coenzyme A ligase and isocitrate dehydrogenase1 are differential proteins upregulated after treated with 300ppm and 600ppm of oleuropein. It seems that Nano Oleuropein is a booster of Krebs cycle with upregulation of Fumarylacetoacetase, succinate-CoA ligase, and isocitrate dehydrogenase1. Uncoordinated Overexpression of some Krebs cycle protein can be one of the inhibition mechanisms on the breast cancer cell line under Oleuropein treatment.