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1.
Am J Hum Genet ; 108(1): 115-133, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33308444

RESUMO

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.


Assuntos
Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Deficiências do Desenvolvimento/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL
2.
J Hum Genet ; 65(2): 193-197, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31767933

RESUMO

Biallelic pathogenic variants in POC1A are ultra rare. They have been reported in 13 families as causing either Short stature, Onychodysplasia, Facial dysmorphism, and hypoTrichosis (SOFT) syndrome, or a milder partially overlapping phenotype, variant POC1A-related syndrome. This pleiotropic effect is likely precipitated by the variant's location and respective affected protein domain. Here, we describe seven patients from two consanguineous Omani families with classic SOFT syndrome and a novel homozygous POC1A variant (c.64G>T; p.(Val22Phe)), which is the first one described for the alternative exon 2. This result refines the POC1A mutational spectrum relevant for exertion of the described pleiotropic effect. Furthermore, six of our patients experienced recurrent mild to severe respiratory difficulties that have not been previously reported for SOFT syndrome and may be an underdiagnosed or a genotype-specific complication that warrants attention in future studies. Thus, our study unravels new aspects of the genotype-phenotype correlation suggested by previous reports.


Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ciclo Celular/genética , Anormalidades Craniofaciais/genética , Proteínas do Citoesqueleto/genética , Nanismo/genética , Estudos de Associação Genética , Hipotricose/genética , Atrofia Muscular/genética , Processamento Alternativo , Criança , Pré-Escolar , Consanguinidade , Éxons/genética , Feminino , Genótipo , Humanos , Masculino , Mutação , Fenótipo
3.
J Inherit Metab Dis ; 42(5): 839-849, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31111503

RESUMO

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/patologia , Erros Inatos do Metabolismo dos Carboidratos/patologia , Domínio Catalítico/genética , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/genética , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Animais , Comportamento Animal , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Modelos Animais de Doenças , Estabilidade Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Multimerização Proteica
4.
BMC Med Genet ; 19(1): 85, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29801475

RESUMO

BACKGROUND: Dyskeratosis congenita (DC) is a rare genetic disorder of bone marrow failure inherited in an X-linked, autosomal dominant or autosomal recessive pattern. It has a wide array of clinical features and patients may be cared for by many medical sub specialties. The typical clinical features consist of lacy reticular skin pigmentation, nail dystrophy and oral leukoplakia. As the disease advances, patients may develop progressive bone marrow failure, pulmonary fibrosis, oesophageal stenosis, urethral stenosis, liver cirrhosis as well as haematological and solid malignancies. Several genes have been implicated in the pathogenesis of dyskeratosis congenita, with the dyskerin pseudouridine synthase 1 (DKC1) gene mutations being the X-linked recessive gene. CASE PRESENTATION: Herein, we report a 31-year-old male with history of recurrent febrile episodes who was found to have reticulate skin pigmentation interspersed with hypopigmented macules involving the face, neck and extremities, hyperkeratosis of palms and soles, nail dystrophy, leukoplakia of the tongue, premature graying of hair, watery eyes and dental caries. Several of his male relatives, including two maternal uncles and three maternal cousins were affected with a similar type of disease condition. Pedigree analysis suggested a possible X-linked pattern of inheritance. Genetic testing in the proband showed a novel hemizygous, non-synonymous likely pathogenic variant [NM_001363.4: c.1054A > G: p.Thr352Ala] in the PUA domain of the DKC1 gene. Quantitative polymerase chain reaction for relative telomere length measurements performed in the proband showed that he had very short telomeres [0.38, compared to a control median of 0.71 (range 0.44-1.19)], which is consistent with the DC diagnosis. Co-segregation analysis of the novel mutation and telomere length measurements in the extended family members could not be performed as they were unwilling to provide consent for testing. CONCLUSIONS: The novel variant detected in the DKC1 gene adds further to the existing scientific literature on the genotype-phenotype correlation of DC, and has important implications for the clinical and molecular characterization of the disease.


Assuntos
Proteínas de Ciclo Celular/genética , Disceratose Congênita/genética , Hemizigoto , Proteínas Nucleares/genética , Mutação Puntual , Adulto , Proteínas de Ciclo Celular/química , Humanos , Masculino , Proteínas Nucleares/química , Linhagem , Domínios Proteicos , Análise de Sequência de DNA , Homeostase do Telômero
5.
Trends Biochem Sci ; 35(4): 220-7, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20060301

RESUMO

The metabolic network has a modular architecture, is robust to perturbations, and responds to biological stimuli and environmental conditions. Through monitoring by metabolite responsive macromolecules, metabolic pathways interact with the transcriptome and proteome. Whereas pathway interconnecting cofactors and substrates report on the overall state of the network, specialised intermediates measure the activity of individual functional units. Transitions in the network affect many of these regulatory metabolites, facilitating the parallel regulation of the timing and control of diverse biological processes. The metabolic network controls its own balance, chromatin structure and the biosynthesis of molecular cofactors; moreover, metabolic shifts are crucial in the response to oxidative stress and play a regulatory role in cancer.


Assuntos
Redes e Vias Metabólicas/fisiologia , Metabolismo dos Carboidratos , Redes e Vias Metabólicas/genética , Transcrição Gênica
6.
Biomolecules ; 14(2)2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38397443

RESUMO

Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the cellular anti-oxidant defense mechanisms, plays a critical role in the pathogenesis of various human diseases. Redox metabolism, comprising a network of enzymes and genes, serves as a crucial regulator of ROS levels and maintains cellular homeostasis. This review provides an overview of the most important human genes encoding for proteins involved in ROS generation, ROS detoxification, and production of reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the genetic disorders that lead to dysregulation of these vital processes. Insights gained from studies on inherited monogenic metabolic diseases provide valuable basic understanding of redox metabolism and signaling, and they also help to unravel the underlying pathomechanisms that contribute to prevalent chronic disorders like cardiovascular disease, neurodegeneration, and cancer.


Assuntos
Antioxidantes , Estresse Oxidativo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/fisiologia , Oxirredução , Antioxidantes/metabolismo , Transdução de Sinais
7.
J Clin Endocrinol Metab ; 108(8): 2087-2098, 2023 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-36658456

RESUMO

CONTEXT: Humans respond profoundly to changes in diet, while nutrition and environment have a great impact on population health. It is therefore important to deeply characterize the human nutritional responses. OBJECTIVE: Endocrine parameters and the metabolome of human plasma are rapidly responding to acute nutritional interventions such as caloric restriction or a glucose challenge. It is less well understood whether the plasma proteome would be equally dynamic, and whether it could be a source of corresponding biomarkers. METHODS: We used high-throughput mass spectrometry to determine changes in the plasma proteome of i) 10 healthy, young, male individuals in response to 2 days of acute caloric restriction followed by refeeding; ii) 200 individuals of the Ely epidemiological study before and after a glucose tolerance test at 4 time points (0, 30, 60, 120 minutes); and iii) 200 random individuals from the Generation Scotland study. We compared the proteomic changes detected with metabolome data and endocrine parameters. RESULTS: Both caloric restriction and the glucose challenge substantially impacted the plasma proteome. Proteins responded across individuals or in an individual-specific manner. We identified nutrient-responsive plasma proteins that correlate with changes in the metabolome, as well as with endocrine parameters. In particular, our study highlights the role of apolipoprotein C1 (APOC1), a small, understudied apolipoprotein that was affected by caloric restriction and dominated the response to glucose consumption and differed in abundance between individuals with and without type 2 diabetes. CONCLUSION: Our study identifies APOC1 as a dominant nutritional responder in humans and highlights the interdependency of acute nutritional response proteins and the endocrine system.


Assuntos
Diabetes Mellitus Tipo 2 , Proteoma , Humanos , Masculino , Proteômica , Glucose , Restrição Calórica
9.
Elife ; 112022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35900202

RESUMO

Interpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species, Raphanus sativus. In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa, Helianthus annuus, or Andropogon gerardi, more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications.


Assuntos
Adenina , Citosina , 5-Metilcitosina/metabolismo , Adenina/metabolismo , Animais , Citosina/química , DNA/metabolismo , Metilação de DNA , Eucariotos/genética , Mamíferos/genética
10.
PLOS Digit Health ; 1(1): e0000007, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36812516

RESUMO

Global healthcare systems are challenged by the COVID-19 pandemic. There is a need to optimize allocation of treatment and resources in intensive care, as clinically established risk assessments such as SOFA and APACHE II scores show only limited performance for predicting the survival of severely ill COVID-19 patients. Additional tools are also needed to monitor treatment, including experimental therapies in clinical trials. Comprehensively capturing human physiology, we speculated that proteomics in combination with new data-driven analysis strategies could produce a new generation of prognostic discriminators. We studied two independent cohorts of patients with severe COVID-19 who required intensive care and invasive mechanical ventilation. SOFA score, Charlson comorbidity index, and APACHE II score showed limited performance in predicting the COVID-19 outcome. Instead, the quantification of 321 plasma protein groups at 349 timepoints in 50 critically ill patients receiving invasive mechanical ventilation revealed 14 proteins that showed trajectories different between survivors and non-survivors. A predictor trained on proteomic measurements obtained at the first time point at maximum treatment level (i.e. WHO grade 7), which was weeks before the outcome, achieved accurate classification of survivors (AUROC 0.81). We tested the established predictor on an independent validation cohort (AUROC 1.0). The majority of proteins with high relevance in the prediction model belong to the coagulation system and complement cascade. Our study demonstrates that plasma proteomics can give rise to prognostic predictors substantially outperforming current prognostic markers in intensive care.

11.
Cell Syst ; 12(8): 780-794.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34139154

RESUMO

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.


Assuntos
Biomarcadores/análise , COVID-19/patologia , Progressão da Doença , Proteoma/fisiologia , Fatores Etários , Contagem de Células Sanguíneas , Gasometria , Ativação Enzimática , Humanos , Inflamação/patologia , Aprendizado de Máquina , Prognóstico , Proteômica , SARS-CoV-2/imunologia
12.
Eur J Hum Genet ; 26(4): 592-598, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29449720

RESUMO

Congenital neurological disorders are genetically highly heterogeneous. Rare forms of hereditary neurological disorders are still difficult to be adequately diagnosed. Pertinent studies, especially when reporting only single families, need independent confirmation. We present three unrelated families in which whole-exome sequencing identified the homozygous non-sense variants c.430[C>T];[C>T] p.(Arg144*), c.1219[C>T];[C>T] p.(Gln407*) and c.1408[C>T];[C>T] p.(Arg470*) in GTPBP2. Their clinical presentations include early onset and apparently non-progressive motor and cognitive impairment, and thereby overlap with findings in a recently described family harbouring a homozygous GTPBP2 splice site variant. Notable differences include structural brain abnormalities (e.g., agenesis of the corpus callosum, exclusive to our patients), and evidence for brain iron accumulation (exclusive to the previously described family). This report confirms pathogenicity of biallelic GTPBP2 inactivation and broadens the phenotypic spectrum. It also underlines that a potential involvement of brain iron accumulation needs clarification. Further patients will have to be identified and characterised in order to fully define the core features of GTPBP2-associated neurological disorder, but future approaches to molecular diagnosis of neurodevelopmental disorders should implement GTPBP2.


Assuntos
Agenesia do Corpo Caloso/genética , Deficiência Intelectual/genética , Sobrecarga de Ferro/genética , Mutação com Perda de Função , Proteínas Monoméricas de Ligação ao GTP/genética , Agenesia do Corpo Caloso/patologia , Alelos , Criança , Feminino , Proteínas de Ligação ao GTP , Humanos , Deficiência Intelectual/patologia , Sobrecarga de Ferro/patologia , Masculino , Fenótipo , Síndrome
13.
Biol Rev Camb Philos Soc ; 90(3): 927-63, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25243985

RESUMO

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner-Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the 'Warburg effect' of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.


Assuntos
Metabolismo/fisiologia , Via de Pentose Fosfato/fisiologia , Humanos , Doenças Metabólicas/fisiopatologia , Via de Pentose Fosfato/genética
14.
Open Biol ; 4: 130232, 2014 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-24598263

RESUMO

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.


Assuntos
Glicólise , Fosfoenolpiruvato/metabolismo , Triose-Fosfato Isomerase/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Gliceraldeído 3-Fosfato/química , Gliceraldeído 3-Fosfato/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Fosfoenolpiruvato/química , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética
15.
J Invest Dermatol ; 134(11): 2823-2832, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24999593

RESUMO

As the outermost barrier of the body, the skin is exposed to multiple environmental factors, including temperature, humidity, mechanical stress, and chemical stimuli such as odorants that are often used in cosmetic articles. Keratinocytes, the major cell type of the epidermal layer, express a variety of different sensory receptors that enable them to react to various environmental stimuli and process information in the skin. Here we report the identification of a novel type of chemoreceptors in human keratinocytes, the olfactory receptors (ORs). We cloned and functionally expressed the cutaneous OR, OR2AT4, and identified Sandalore, a synthetic sandalwood odorant, as an agonist of this receptor. Sandalore induces strong Ca(2+) signals in cultured human keratinocytes, which are mediated by OR2AT4, as demonstrated by receptor knockdown experiments using RNA interference. The activation of OR2AT4 induces a cAMP-dependent pathway and phosphorylation of extracellular signal-regulated kinases (Erk1/2) and p38 mitogen-activated protein kinases (p38 MAPK). Moreover, the long-term stimulation of keratinocytes with Sandalore positively affected cell proliferation and migration, and regeneration of keratinocyte monolayers in an in vitro wound scratch assay. These findings combined with our studies on human skin organ cultures strongly indicate that the OR 2AT4 is involved in human keratinocyte re-epithelialization during wound-healing processes.


Assuntos
Regulação da Expressão Gênica , Queratinócitos/citologia , Queratinócitos/metabolismo , Receptores Odorantes/metabolismo , Santalum/química , Cicatrização , Cálcio/química , Movimento Celular , Proliferação de Células , Clonagem Molecular , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Odorantes , Neurônios Receptores Olfatórios/metabolismo , Interferência de RNA , Transdução de Sinais , Pele/metabolismo
16.
Front Oncol ; 2: 212, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23346549

RESUMO

Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

17.
Oncotarget ; 3(11): 1356-69, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23154538

RESUMO

The glycolytic enzyme pyruvate kinase (PK) is required for cancer development, and has been implicated in the metabolic transition from oxidative to fermentative metabolism, the Warburg effect. However, the global metabolic response that follows changes in PK activity is not yet fully understood. Using shotgun proteomics, we identified 31 yeast proteins that were regulated in a PK-dependent manner. Selective reaction monitoring confirmed that their expression was dependent on PK isoform, level and activity. Most of the PK targets were amino acid metabolizing enzymes or factors of protein translation, indicating that PK plays a global regulatory role in biosynthethic amino acid metabolism. Indeed, we found strongly altered amino acid profiles when PK levels were changed. Low PK levels increased the cellular glutamine and glutamate concentrations, but decreased the levels of seven amino acids including serine and histidine. To test for evolutionary conservation of this PK function, we quantified orthologues of the identified PK targets in thyroid follicular adenoma, a tumor characterized by high PK levels and low respiratory activity. Aminopeptidase AAP-1 and serine hydroxymethyltransferase SHMT1 both showed PKM2- concentration dependence, and were upregulated in the tumor. Thus, PK expression levels and activity were important for maintaining cellular amino acid homeostasis. Mediating between energy production, ROS clearance and amino acid biosynthesis, PK thus plays a central regulatory role in the metabolism of proliferating cells.


Assuntos
Adenoma/enzimologia , Aminoácidos/metabolismo , Piruvato Quinase/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Adenoma/genética , Adenoma/patologia , Aminopeptidases/metabolismo , Processos de Crescimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Glicina Hidroximetiltransferase/metabolismo , Homeostase , Humanos , Isoenzimas/metabolismo , Proteoma/metabolismo , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Regulação para Cima
18.
Cell Metab ; 14(3): 415-27, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21907146

RESUMO

In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in yeast, we discovered that central metabolism is self-adapting to synchronize redox metabolism when respiration is activated. Low PYK activity activated yeast respiration. However, levels of reactive oxygen species (ROS) did not increase, and cells gained resistance to oxidants. This adaptation was attributable to accumulation of the PYK substrate phosphoenolpyruvate (PEP). PEP acted as feedback inhibitor of the glycolytic enzyme triosephosphate isomerase (TPI). TPI inhibition stimulated the pentose phosphate pathway, increased antioxidative metabolism, and prevented ROS accumulation. Thus, a metabolic feedback loop, initiated by PYK, mediated by its substrate and acting on TPI, stimulates redox metabolism in respiring cells. Originating from a single catalytic step, this autonomous reconfiguration of central carbon metabolism prevents oxidative stress upon shifts between fermentation and respiration.


Assuntos
Respiração Celular/fisiologia , Retroalimentação Fisiológica , Glicólise/fisiologia , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Triose-Fosfato Isomerase/metabolismo , Proliferação de Células , Cromatografia Líquida , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Oxirredução , Estresse Oxidativo/genética , Via de Pentose Fosfato , Reação em Cadeia da Polimerase , Piruvato Quinase/genética , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Triose-Fosfato Isomerase/genética
19.
Oncotarget ; 2(5): 393-400, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21789790

RESUMO

The Warburg effect describes the circumstance that tumor cells preferentially use glycolysis rather than oxidative phosphorylation for energy production. It has been reported that this metabolic reconfiguration originates from a switch in the expression of alternative splice forms (PKM1 and PKM2) of the glycolytic enzyme pyruvate kinase (PK), which is also important for malignant transformation.However, analytical evidence for this assumption was still lacking. Using mass spectrometry, we performed an absolute quantification of PKM1 and PKM2 splice isoforms in 25 human malignant cancers, 6 benign oncocytomas, tissue matched controls, and several cell lines. PKM2 was the prominent isoform in all analyzed cancer samples and cell lines. However, this PKM2 dominance was not a result of a change in isoform expression, since PKM2 was also the predominant PKM isoform in matched control tissues. In unaffected kidney, lung, liver, and thyroid, PKM2 accounted for a minimum of 93% of total PKM, for 80% - 96% of PKM in colon,and 55% - 61% of PKM in bladder. Similar results were obtained for a panel of tumor and non-transformed cell lines, where PKM2 was the predominant form.Thus, our results reveal that an exchange in PKM1 to PKM2 isoform expression during cancer formation is not occurring, nor do these results support conclusions that PKM2 is specific for proliferating, and PKM1 for non-proliferating tissue.


Assuntos
Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Neoplasias/enzimologia , Isoformas de Proteínas/metabolismo , Piruvato Quinase/metabolismo , Processamento Alternativo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Glicólise/genética , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Espectrometria de Massas , Neoplasias/genética , Fosforilação Oxidativa , Isoformas de Proteínas/genética , Piruvato Quinase/genética , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia
20.
Antioxid Redox Signal ; 15(2): 311-24, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21348809

RESUMO

AIMS: A shift in primary carbon metabolism is the fastest response to oxidative stress. Induced within seconds, it precedes transcriptional regulation, and produces reducing equivalents in form of NADPH within the pentose phosphate pathway (PPP). RESULTS: Here, we provide evidence for a regulatory signaling function of this metabolic transition in yeast. Several PPP-deficiencies caused abnormal accumulation of intermediate metabolites during the stress response. These PPP-deficient strains had strong growth deficits on media containing oxidants, but we observed that part of their oxidant-phenotypes were not attributable to the production of NADPH equivalents. This pointed to a second, yet unknown role of the PPP in the antioxidant response. Comparing transcriptome profiles obtained by RNA sequencing, we found gene expression profiles that resembled oxidative conditions when PPP activity was increased. Vice versa, deletion of PPP enzymes disturbed and delayed mRNA and protein expression during the antioxidant response. INNOVATION: Thus, the transient activation of the PPP is a metabolic signal required for balancing and timing gene expression upon an oxidative burst. CONCLUSION: Consequently, dynamic rearrangements in central carbon metabolism seem to be of major importance for eukaryotic redox sensing, and represent a novel class of dynamic gene expression regulators.


Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica , Via de Pentose Fosfato , Transcrição Gênica , Transporte de Elétrons , Perfilação da Expressão Gênica , Glicólise , Mutação , NADP/metabolismo , Oxirredução
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