RESUMEN
Regeneration is rare in mammals, but spiny mice (Acomys spp.) naturally regenerate skin and ear holes. Inflammation is thought to inhibit regeneration during wound healing, but aspects of inflammation contribute to both regeneration and pathogen defense. We compared neutrophil traits among uninjured, regeneration-competent (Acomys: A. cahirinus, A. kempi, A. percivali) and -incompetent (Mus musculus: Swiss Webster, wild-caught strains) murids to test for constitutive differences in neutrophil quantity and function between these groups. Neutrophil quantity differed significantly among species. In blood, Acomys had lower percentages of circulating neutrophils than Mus; and in bone marrow, Acomys had higher percentages of band neutrophils and lower percentages of segmented neutrophils. Functionally, Acomys and Mus neutrophils did not differ in their ability to migrate or produce reactive oxygen species, but Acomys neutrophils phagocytosed more fungal zymosan. Despite this enhanced phagocytosis activity, Acomys neutrophils were not more effective than Mus neutrophils at killing Escherichia coli. Interestingly, whole blood bacteria killing was dominated by serum in Acomys versus neutrophils only or neutrophils and serum in Mus, suggesting that Acomys primarily rely on serum to kill bacteria whereas Mus do not. These subtle differences in neutrophil traits may allow regeneration-competent species to offset damaging effects of inflammation without compromising pathogen defense.
Asunto(s)
Ratones/sangre , Murinae/sangre , Neutrófilos/fisiología , Regeneración , Animales , Especificidad de la EspecieRESUMEN
Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. MMR recognizes and repairs DNA mismatches and small insertion/deletion loops. Carriers of MMR gene variants have a high risk of developing colorectal, endometrial, ovarian, and other extracolonic carcinomas. We report on an ovarian cancer patient who carries a germline MSH2 c.1A>C variant which alters the translation initiation codon. Mutations affecting the MSH2 start codon have been described previously for LS-related malignancies. However, the patients often lack a clear family history indicative of LS and their tumors often fail to display microsatellite instability, a hallmark feature of LS. Therefore, the pathogenicity of start codon variants remains undefined. Loss of the MSH2 start codon has been predicted to result in a truncated protein translated from a downstream in-frame AUG that would lack the first 25 amino acids. We therefore purified recombinant MSH2(NΔ25)-MSH6 and MSH2(NΔ25)-MSH3 to examine their DNA lesion recognition and adenosine nucleotide processing functions in vitro. We found that the MSH2(NΔ25) mutant confers distinct biochemical defects on MSH2-MSH6, but does not have a significant effect on MSH2-MSH3. We confirmed that expression of the MSH2 c.1A>C cDNA results in the production of multiple protein products in human cells that may include the truncated and full-length forms of MSH2. An in vivo MMR assay revealed a slight reduction in MMR efficiency in these cells. These data suggest that mutation of the MSH2 initiation codon, while not a strong, high-risk disease allele, may have a moderate impact on disease phenotype.
Asunto(s)
Codón Iniciador/genética , Proteínas de Unión al ADN/genética , Proteína 2 Homóloga a MutS/genética , Mutación , Neoplasias Ováricas/genética , Secuencia de Aminoácidos , Disparidad de Par Base , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Salud de la Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Linaje , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for â¼8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.
Asunto(s)
Adenosina Trifosfato/química , Proteínas de Unión al ADN/química , Complejos Multienzimáticos/química , Proteína 2 Homóloga a MutS/química , Adenosina Difosfato/química , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Neoplasias Colorrectales Hereditarias sin Poliposis/enzimología , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Hidrólisis , Cinética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismoRESUMEN
Hereditary nonpolyposis colorectal cancer is caused by germline mutations in DNA mismatch repair genes. The majority of cases are associated with mutations in hMSH2 or hMLH1; however, about 12% of cases are associated with alterations in hMSH6. The hMSH6 protein forms a heterodimer with hMSH2 that is capable of recognizing a DNA mismatch. The heterodimer then utilizes its adenosine nucleotide processing ability in an, as of yet, unclear mechanism to facilitate communication between the mismatch and a distant strand discrimination site. The majority of reported mutations in hMSH6 are deletions or truncations that entirely eliminate the function of the protein; however, nearly a third of the reported variations are missense mutations whose functional significance is unclear. We analyzed seven cancer-associated single amino acid alterations in hMSH6 distributed throughout the functional domains of the protein to determine their effect on the biochemical activity of the hMSH2-hMSH6 heterodimer. Five alterations affect mismatch-stimulated ATP hydrolysis activity providing functional evidence that missense variants of hMSH6 can disrupt mismatch repair function and may contribute to disease. Of the five mutants that affect mismatch-stimulated ATP hydrolysis, only two (R976H and H1248D) affect mismatch recognition. Thus, three of the mutants (G566R, V878A, and D803G) appear to uncouple the mismatch binding and ATP hydrolysis activities of the heterodimer. We also demonstrate that these three mutations alter ATP-dependent conformation changes of hMSH2-hMSH6, suggesting that cancer-associated mutations in hMSH6 can disrupt the intramolecular signaling that coordinates mismatch binding with adenosine nucleotide processing.
Asunto(s)
Adenosina Trifosfato/metabolismo , Disparidad de Par Base/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/enzimología , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Mutación Missense/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Hidrólisis , Modelos Moleculares , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Aberrant crypt foci (ACF) are microscopic surface abnormalities that are putative precursors to colorectal cancer (CRC). ACF exhibit similar histological and molecular abnormalities to adenomas and CRC and potentially represent useful biomarkers of cancer risk. Microsatellite instability (MSI) is one molecular abnormality identified in concurrent ACF from CRC patients that may indicate a risk for progression. To determine if MSI can be detected in ACF from cancer-free subjects, we examined 45 ACF from 20 subjects undergoing colonoscopies. The group included 12 patients at elevated risk for CRC based on family history of CRC or personal history of CRC or advanced adenoma and 8 patients with no known risk factors. ACF were identified using close-focus magnifying chromendoscopy and collected by biopsy in situ. Genomic DNA was prepared from ACF and adjacent normal colonic epithelium isolated by laser capture microdissection and analyzed for MSI. MSI was identified in at least one marker from 9 of 30 (30%) lesions from patients at elevated risk for CRC and in 2 of 15 (13%) lesions from average risk patients. Using methylation-specific PCR analysis, we also examined the ACF for promoter hypermethylation of the DNA repair genes hMLH1 and MGMT and found moderate changes (8/39 and 3/32, respectively). Although we found only a limited relationship between hMLH1 hypermethylation and MSI, all the lesions with MGMT hypermethylation displayed an MSI-low phenotype. These lesions may be precursors to MSI-low CRC, providing a potential early biomarker to assess the effects of cancer prevention strategies.