Mechanistic differences in RNA-dependent DNA polymerization and fidelity between murine leukemia virus and HIV-1 reverse transcriptases

We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (Kd) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (kpol). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the Kd and kpol values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Clinical and genetic profile of patients with medullary thyroid cancer treated in the Cancer Centre - Institute of Oncology in Warsaw

Wstęp: Celem pracy jest analiza wyników badań genetycznych: rozkładu lokalizacji i częstości mutacji oraz ujawnienie korelacji prezentowanych klinicznie fenotypów u chorych z rakiem rdzeniastym tarczycy (MTC, medullary thyroid carcinoma), wykazanie różnic między postacią sporadyczną i dziedziczną, a także określenie odrębności występujących w grupie chorych pozostających pod stałą obserwacją ośrodka autorów niniejszej pracy. Materiał i metody: W Centrum Onkologii w Warszawie w latach 1997-2005, leczono 212 chorych z rakiem rdzeniastym tarczycy. U większości z nich wykonano badanie genetyczne DNA uzyskanego z leukocytów krwi obwodowej w celu zidentyfikowania mutacji genu RET i ustalenia postaci MTC. Badaniu DNA poddano także krewnych chorych z rodzinnym rakiem rdzeniastym tarczycy, aby wyodrębnić bezobjawowych nosicieli od osób nieobciążonych patogenną mutacją. Wyniki: W grupie chorych, u których zakończono analizę genetyczną, mutacje w genie RET stwierdzono w 46 przypadkach (22%). U pozostałych chorych nie ujawniono patogennej mutacji i rozpoznano sporadycznego MTC. Zespół mnogiej gruczolakowatości wewnątrzwydzielniczej typu 2A i rodzinnego typu MTC (MEN 2A/FMTC, multiple endocrine neoplasia type 2A/familial type of MTC syndrome) rozpoznano u 44 pacjentów, natomiast zespół mnogiej gruczolakowatości wewnątrzwydzielniczej typu 2B (MEN 2B, multiple endocrine neoplasia type 2B) - u 2 osób. W trakcie badania porównano wiek rozpoznania i wieloogniskowość raka u chorych ze sporadycznym i rodzinnym MTC. Uzyskane wyniki korelują z danymi z innych ośrodków. Natomiast, rozkład lokalizacji mutacji i ich częstość, a także niektóre dane kliniczne, takie jak częstość ujawnienia guza chromochłonnego nadnerczy jako pierwszej patologii z zespołu wielogruczołowego, różnią się od prezentowanych w literaturze i wymagają znalezienia przyczyn. Wnioski: Badanie mutacji genu RET jest wiarygodnym narzędziem diagnostycznym i należy je traktować jako badanie przesiewowe u wszystkich chorych z MTC i innymi składowymi zespołu wielogruczołowego.Introduction: The aim of this study was to analyse the distribution and frequency of mutations and their correlations with clinical phenotypes of patients with MTC, to reveal the differences between sporadic and familial type of MTC, and to describe the phenotypes of patients. Materials and methods: 212 patients with medullary thyroid cancer (MTC) were treated in Cancer Centre in Warsaw between 1997 and 2005. In most patients, DNA isolated from peripheral blood leukocytes was tested for RET gene mutations by sequencing and accordingly MTC form was assessed. Genetic testing was performed in the relatives of patients with familial MTC in order to distinguish asymptomatic mutation carriers from noncarriers. Results: RET gene mutations were identified in 46 patients (22%). The others were found noncarriers and sporadic MTC was diagnosed. MEN 2A/FMTC syndrome (multiple endocrine neoplasia type 2A/ familial type of MTC) was diagnosed in 44 patients, MEN 2B syndrome (multiple endocrine neoplasia type 2B) in 2 patients. In patients with sporadic and familial MTC, age at diagnosis and multifocal occurrence was analysed, and the results were found to be in accordance with those of other research centres. However, the distribution and frequency of mutations, as well as some clinical data, such as the frequency of pheochromocytoma occurrence as the first manifestation of MEN syndrome, differed from the published data, and further studies are necessary to reveal the reasons of these differences. Conclusions: DNA testing for RET gene mutations is reliable as a diagnostic tool and therefore it should be performed for screening of all patients with MTC or other diseases of MEN syndrome

Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs

The Frequency of Selected Polymorphic Variants of the RET Gene in Patients with Medullary Thyroid Carcinoma and in the General Population of Central Poland.

The object of this work was to compare the frequency of three polymorphic changes in the RET proto-oncogene: L769L, S836S, and S904S in patients with medullary thyroid carcinoma (MTC; n = 246) and in the general population (n = 420 for single-nucleotide polymorphism [SNP] L769L and S904S; n = 411 for SNP 836). We tried to investigate how the harbored SNPs affect the age at onset of sporadic medullary thyroid carcinoma (sMTC) and MTC in carriers of known pathogenic mutations at codons 634 and 791 of the RET gene. A statistically significant difference was found in the frequency of the heterozygous change L769L in patients with sMTC (48.3%) and in unaffected individuals (39.5%). The presence of the polymorphic change L769L in the RET gene predisposes to the development of sMTC and also lowers the age of onset of MTC in carriers of the homozygous polymorphic variant L769L. The presence of this polymorphic change in MTC patients carrying, at the same time, the RET codon 634 mutation lowers the age of onset of MTC in this group