Searching for regulators of the mammalian dNTP pool

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular: Fecha de lectura: 23-10-2015DNA replication is a tightly controlled process with its misregulation leading to a type of DNA damage, known as replication stress (RS). RS is essentially defined as an accumulation of unprotected single-stranded DNA (ssDNA) at stalled replication forks (RF). Due to the recombinogenic nature of ssDNA, it is a cause of genomic rearrangements frequently observed in cancer. One way by which ssDNA can arise is an insufficient supply of nucleotides (dNTPs), which limits the progression of the DNA polymerases. Accordingly, reduced dNTP levels have been proposed as a source of genomic instability in cancer. In order to protect their genomes, cells need to detect and limit the amount of ssDNA. ATR is the initial kinase that responds to RS in mammals, resulting in a phosphorylation cascade that finally leads to cell cycle arrest and safeguarding of replication fork integrity. While it remains unclear how exactly ATR suppresses RS, evidences from yeast suggest that ATR might reduce RS through stimulating the production of nucleotides. In Saccharomyces cerevisiae (S.cerevisiae), lethality of the ATR ortholog Mec1 can be rescued by concomitant deletion of Suppressor of Mec1 lethality1 (Sml1), an inhibitor of the ribonucleotide reductase (RNR). In all eukaryotes, RNR catalyzes a rate limiting step in dNTP synthesis by reducing NDPs to dNDPs in a tightly controlled process. The RNR is a heterotetrameric complex composed of a catalytic subunit (Rnr1 and RRM1 in yeast and mouse respectively) and a regulatory subunit (Rnr2 and RRM2 in yeast and mosue respectively) whose structure and function are well conserved throughout evolution. Although the sequence where Sml1 binds to Rnr1 is conserved in mammals and yeast Sml1 binds and inhibits the mammalian RNR, it remains unclear whether ATR-dependent regulation of RNR is conserved beyond yeast. In this work we investigated the putative relationship between RS, ATR and RNR activity, and searched for mammalian regulators of the RNR. Although we failed to identify new regulators of the RNR, we could establish that increased RNR activity alleviates phenotypes associated with ATR-deficiency in mice in vitro and in vivo, pointing towards a functional conservation of the ATR-dependent regulation of the dNTP-pool in mammals. In addition, we show that a point mutation within a single conserved residue in RRM1 prevents binding to RRM2 causing early embryonic lethality in mice. Despite the impact of the mutation, Rrm1+/WG mice present no obvious phenotype suggesting that RRM1 exists in excess in mammalian cells. This data reveals that binding of RRM1 to RRM2 is essential for viability, and provides the first loss-of-function model of the RNR complex for genetic studies in mammalsLa replicación del ADN es un proceso que ha de ser controlado con precisión. Su desregulación genera un tipo de daño en el ADN conocido como estrés replicativo (ER). El ER se define como la acumulación de ADN de cadena sencilla (ssDNA) desprotegido en las horquillas de replicación (RF) que se han bloqueado. Debido a su naturaleza recombinogénica, el ssDNA puede causar reordenamientos genómicos, algunos de los cuales ocurren frecuentemente en cáncer. Una de las razones por las que se acumula ssDNA es un aporte insuficiente de nucleótidos (dNTPs) que limita la progresión de las ADN-polimerasas. De este modo, se ha propuesto que los niveles reducidos de dNTPs son una fuente de inestabilidad genómica en cáncer. Con objeto de proteger sus genomas, las células necesitan detectar y limitar la cantidad de ssDNA. ATR es la principal quinasa que responde a ER en mamíferos, lo que resulta en la activación de una cascada de fosforilaciones que limita el avance en el ciclo celular al tiempo que mantiene la integridad de las horquillas de replicación. Si bien no se conoce el mecanismo exacto por el que la activación de ATR suprime ER, los resultados obtenidos en levadura sugieren que ATR podría estimular la producción de nucleótidos y así limitar el ER. En Saccharomyces cerevisiae, la mutación del ortólogo de ATR, Mec1, es letal y este fenotipo puede ser rescatado mediante la deleción simultánea de un inhibidor de la enzima ribonucleótido reductasa (RNR), Suppressor of Mec1 lethality 1 (Sml1). En todos los organismos eucariotas la RNR cataliza un paso limitante en la síntesis de dNTPs, reduciendo NDPs a dNDPs en un proceso que está altamente regulado. La RNR es un complejo heterotetramérico compuesto por una subunidad catalítica (Rnr1 o RRM1; en levadura o ratón, respectivamente) y una subunidad reguladora (Rnr2 o RRM2; en levadura o ratón, respectivamente), cuya estructura y función están muy conservadas a lo largo de la evolución. A pesar de que la secuencia que Sml1 reconoce en Rnr1 está conservada desde levaduras a mamíferos, y a pesar de que Sml1 de levadura une e inhibe a la RNR de mamíferos in vitro, se desconoce si la regulación de la RNR por parte de ATR se conserva más allá de levaduras. En este trabajo hemos investigado la posible relación entre ER, ATR y la actividad RNR, y hemos buscado nuevos reguladores de la RNR en mamíferos. Si bien no hemos conseguido identificar nuevos reguladores de la RNR, hemos establecido que un incremento de la actividad RNR alivia los fenotipos ocasionados por una deficiencia en ATR in vitro e in vivo, lo que indica que la regulación de los reservorios de dNTPs por parte de ATR está conservada en mamíferos. Además, hemos mostrado que la mutación de un sólo residuo conservado de RRM1 evita la interacción de esta proteína con RRM2, y causa letalidad embrionaria temprana en ratones. A pesar del impacto de esta mutación, los ratones Rrm1+/WG no presentan un fenotipo obvio, lo que sugiere que RRM1 está presente en exceso en células de mamífero. Estos datos revelan que la interacción entre RRM1 y RRM2 es esencial para la viabilidad celular, y proveen el primer modelo genético de pérdida de función de la RNR en mamíferosThis work has been funded by the Spanish Ministry of Economics and Competitiveness (FPI scholarship

Adipocyte-derived endotrophin promotes malignant tumor progression

Adipocytes represent a major cell type in the mammary tumor microenvironment and are important for tumor growth. Collagen VI (COL6) is highly expressed in adipose tissue, upregulated in the obese state, and enriched in breast cancer lesions and is a stimulator of mammary tumor growth. Here, we have described a cleavage product of the COL6??3 chain, endotrophin (ETP), which serves as the major mediator of the COL6-mediated tumor effects. ETP augmented fibrosis, angiogenesis, and inflammation through recruitment of macrophages and endothelial cells. Moreover, ETP expression was associated with aggressive mammary tumor growth and high metastatic growth. These effects were partially mediated through enhanced TGF-?? signaling, which contributes to tissue fibrosis and epithelial-mesenchymal transition (EMT) of tumor cells. Our results highlight the crucial role of ETP as an obesity-associated factor that promotes tumor growth in the context of adipocyte interactions with tumor and stromal cells.open302

Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis

AbstractAnti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3

Immunohistochemical localization of collagen VI in diabetic glomeruli

Immunohistochemical localization of collagen VI in diabetic glomeruli. Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the α1(VI)- and α2(VI)-chains and the N-terminal part of α3(VI)-chain, respectively, revealed no difference in staining pattern. Comparison of the immunohistochemical results with clinical parameters of diabetic nephropathy suggested that increasing collagen VI deposition may be an indicator of the irreversible remodeling of the glomerular matrix to nodular GS which is associated with functional insufficiency. Our findings indicate striking differences of the mesangial matrix composition in diffuse and nodular GS. These observations together with earlier results provide evidence for a “switch” in the matrix protein production in association with the development of nodular GS in diabetic nephropathy

Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA

Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA.BackgroundAutoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes.MethodsThis study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA.ResultsThe rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA.ConclusionsThis expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely

Subclassifying ANCA-associated vasculitis: a unifying view of disease spectrum.

This editorial refers to Clinical impact of subgrouping ANCA-associated vasculitis according to antibody specificity beyond the clinicopathological classification, Deshayes et al

Treatment of ANCA-Associated Vasculitis: New Therapies and a Look at Old Entities

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a small-vessel vasculitis that primarily comprises 2 clinical syndromes: granulomatosis with polyangiitis and microscopic polyangiitis. Cyclophosphamide and glucocorticoids have traditionally been used for induction of remission. However, more recent studies have shown that rituximab is as effective as cyclophosphamide for induction therapy in patients with newly diagnosed severe AAV and superior for patients with relapsing AAV. There is also accumulating evidence indicating a potential role of rituximab for maintenance therapy in AAV. In this article, we will review the evidence supporting the various treatment choices for patients with AAV

Clinical, Radiographic, Histopathologic, and Serologic Features and their Differentiation from Wegener Granulomatosis

n/