No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms

G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha- chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G- proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents

Structurally constrained protein evolution: results from a lattice simulation

We simulate the evolution of a protein-like sequence subject to point mutations, imposing conservation of the ground state, thermodynamic stability and fast folding. Our model is aimed at describing neutral evolution of natural proteins. We use a cubic lattice model of the protein structure and test the neutrality conditions by extensive Monte Carlo simulations. We observe that sequence space is traversed by neutral networks, i.e. sets of sequences with the same fold connected by point mutations. Typical pairs of sequences on a neutral network are nearly as different as randomly chosen sequences. The fraction of neutral neighbors has strong sequence to sequence variations, which influence the rate of neutral evolution. In this paper we study the thermodynamic stability of different protein sequences. We relate the high variability of the fraction of neutral mutations to the complex energy landscape within a neutral network, arguing that valleys in this landscape are associated to high values of the neutral mutation rate. We find that when a point mutation produces a sequence with a new ground state, this is likely to have a low stability. Thus we tentatively conjecture that neutral networks of different structures are typically well separated in sequence space. This results indicates that changing significantly a protein structure through a biologically acceptable chain of point mutations is a rare, although possible, event.Comment: added reference, to appear on European Physical Journal

Point mutations and diseases

Mutacije su nasljedne i trajne promjene u strukturi DNA molekule. Mutacije nastaju spontanim greškama tijekom replikacije DNA ili mogu biti inducirane nekim mutagenim agensima. Točkaste mutacije su mutacije koje nastaju supstitucijom jedne baze ili jednog baznog para drugim baznim parom i uzrok su brojnim bolestima. Prva bolest za koju je otkriveno da ju uzrokuje jedna točkasta mutacija je bila anemija srpastih stanica. Ova bolest uzrokovana je jednom točkastom mutacijom u ß globinskom lancu hemoglobina što za posljedicu ima abnormalne, rigidne eritrocite. To ima ozbiljne i često smrtonosne posljedice za bolesnika. No pojava ove mutacije u subtropskim i tropskim područjima dala je evolucijsku prednost osobama koje ju nose. Naime heterozigoti za ovu mutaciju imaju selekcijsku prednost budući da ne oboljevaju od malarije koja je česta upravo u tim područjima. Točkaste mutacije koje zahvaćaju mitohondrijsku DNA nasljeđuju se samo majčinskom linijom i uglavnom zahvaćaju kodirajuću DNA budući da mitohondriji ne sadrže introne kakvi su prisutni u genomu jezgre eukariota. MELAS sindrom i Leberova nasljedna optička neuropatija su primjeri bolesti uzrokovanih točkastim mutacijama u mtDNA. MELAS sindrom je teška i smrtonosna bolest uzrokovana defektnim proteinima koji grade kompleks I u mitohondriju i posebice pogađa tkiva poput mozga i mišića koja najviše ovise o staničnom ATP-u nastalom procesom oksidativne fosforilacije u mitohondrijima. Leberova nasljedna optička neuropatija za posljedicu ima potpuni gubitak vida kod vrlo mladih osoba. Veliki izazov za istraživače danas predstavlja otkrivanje novih metoda za detekciju točkastih mutacija, kao i različitih metoda njihovog liječenja odnosno smanjivanja negativnog učinka po stanicu i organizam primjerice pomoću genske terapije.Mutations are hereditary and permanent changes in DNA structure. Mutations can occur spontaneously during DNA replication or they can be induced by mutagenic agents. Point mutations are a type of mutation that causes the replacement of a single base nucleotide with another nucleotide in DNA and they are responsible for many diseases. Sickle cell anemia was the first disease known to be caused by one point mutation. The cause of this disease is one point mutation in ß globin chain of hemoglobin which leads to formation of abnormal erythrocytes and that often includes severe and lethal consequences for the patient. Occurance of this mutation in tropic and subtropic areas, where malaria is also very common, made persons who were heterozygous for sickle cell anemia better adapted because this way they had much greater chance of surviving malaria. Point mutations that occur in mitochondrial DNA (mtDNA) are inherited only by mother line and since mitochondrion does not have intrones similar to those in nuclear genome of eucaryotes, these mutations usually affect coding region of mtDNA. Examples of such diseases are MELAS syndrome and Leber's hereditary optic neuropathy. MELAS syndrome is a severe and lethal disease caused by defect in proteins that build complex I in mitochondrion. It specially affects brain and muscle tissue which mostly depend on cell's ATP produced during the process of oxidative phosphorylation in mitochondrion. Leber's hereditary optic neuropathy causes complete loss of vision and it affects very young persons. Today researchers aim to find new methods for point mutations detection. They are also trying to discover various other methods of point mutations threatment like gene therapy. These methods help reduce negative effects of mutations on cells and organisms

All-atom simulations reveal how single point mutations promote serpin misfolding

Protein misfolding is implicated in many diseases, including the serpinopathies. For the canonical inhibitory serpin {\alpha}1-antitrypsin (A1AT), mutations can result in protein deficiencies leading to lung disease, and misfolded mutants can accumulate in hepatocytes leading to liver disease. Using all-atom simulations based on the recently developed Bias Functional algorithm we elucidate how wild-type A1AT folds and how the disease-associated S (Glu264Val) and Z (Glu342Lys) mutations lead to misfolding. The deleterious Z mutation disrupts folding at an early stage, while the relatively benign S mutant shows late stage minor misfolding. A number of suppressor mutations ameliorate the effects of the Z mutation and simulations on these mutants help to elucidate the relative roles of steric clashes and electrostatic interactions in Z misfolding. These results demonstrate a striking correlation between atomistic events and disease severity and shine light on the mechanisms driving chains away from their correct folding routes.Comment: Final version. Supplementary Information include

Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles.

Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1NL4-3. HIV-1NL4-3 variants were capable of CD4+ host entry and reverse transcription as WT HIV-1NL4-3, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations

Point mutations of the P53 gene, human hepatocellular carcinoma and aflatoxins

The tumor suppressor p53 exerts important protective functions towards DNA-damaging agents. Its inactivation by allelic deletions or point mutations within the P53 gene as well as complex formation of wildtype p53 with cellular or viral proteins is a common and crucial event in carcinogenesis. Mutations increase the half-life of the p53 protein allowing the immunohistochemical detection and anti-p53 antibody formation. Distinct G to T point mutations in codon 249 leading to a substitution of the basic amino acid arginine by the neutral amino acid serin are responsible for the altered functionality of the mutant gene product and were originally identified in 8 of 16 Chinese and 5 of 10 African HCC patients. Both groups are frequently exposed to mycotoxin contaminations of their food. Today an average P53 gene mutation rate of 25% is assumed for high-aflatoxin B1-exposure regions. This is double the rate observed in low-aflatoxin B1-exposure countries. Although many HCC patients displaying P53 mutations also suffer from HBV infection, which itself can lead to rearrangements of P53 coding regions or induce the synthesis of viral proteins possibly interacting with p53, the specific G to T transversion within codon 249 of the P53 gene seems to directly reflect the extent of aflatoxin B1 exposure