Quaternary structure change as a mechanism for the regulation of thymidine kinase 1-like enzymes

SummaryThe human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp) and the reaction products (TMP/ADP). In combination with fluorescence spectroscopy and mutagenesis experiments, our results demonstrate that ATP binding is linked to a substantial reorganization of the enzyme quaternary structure, leading to a transition from a closed, inactive conformation to an open, catalytic state. We hypothesize that these structural changes are relevant to enzyme function in situ as part of the catalytic cycle and serve an important role in regulating enzyme activity by amplifying the effects of feedback inhibitor binding

Profile of Contributors of University Radio in Spain: The Case of Students in the Area of Information Science

This article examines the reality of Spanish university radio, a relatively recent and poorly studied reality (only 40 years since Radio UNED began its first recordings). University radio in Spain is very heterogeneous and diverse, these features are clearly reflected in one of the parameters of analysis of this phenomenon; the profile of its contributors and / or participants. In this sense, Spanish university radio agglutinates a wide range of possibilities, from broadcasters who make their broadcats available to the university community as a whole (Professor-Research, university staff and students) as well as to the rest of society, building bridges with them and bringing about the participation of anonymous citizens, associations or NGOs. At the other extreme, there are radio stations that only allow the participation of students from the area of information science, configuring them as authentic practical platforms for these students, and they receive additional training that may be essential in order to gain access to the labour market. This has been evident on many occasions, and we have corroborated this in interviews carried out with various partners and those in charge of the stations

Late presentation of human T-lymphotropic virus type 1 infection in Spain reflects suboptimal testing strategies

Diagnosis; Myelopathy; Sexually transmitted infectionsDiagnóstico; Mielopatía; Infecciones de transmisión sexualDiagnòstic; Mielopatia; Infeccions de transmissió sexualObjectives Although only 10% of persons infected with human T-lymphotropic virus type 1 (HTLV-1) may develop virus-associated illnesses over their lifetime, missing the earlier diagnosis of asymptomatic carriers frequently leads to late presentation. Methods A nationwide HTLV-1 register was created in Spain in 1989. We examined the main demographics and clinical features at the time of the first diagnosis for more than three decades. Results A total of 428 individuals infected with HTLV-1 had been reported in Spain until the end of 2021. Up to 96 (22%) individuals presented clinically with HTLV-1-associated conditions, including subacute myelopathy (57%), T-cell lymphoma (34%), or Strongyloides stercoralis infestation (8%). Since 2008, HTLV-1 diagnosis has been made at blood banks (44%) or clinics (56%). Native Spaniards and Sub-Saharan Africans are overrepresented among patients presenting with HTLV-1-associated illnesses suggesting that poor epidemiological and/or clinical suspicion, which led to the late presentation are more frequent in them than carriers from Latin America (LATAM) (31.7% vs 20.4%, respectively; P = 0.015). Conclusion HTLV-1 infection in Spain is frequently diagnosed in patients presenting with characteristic illnesses. Although screening in blood banks mostly identifies asymptomatic carriers from LATAM, a disproportionately high number of Spaniards and Africans are diagnosed too late at the time of clinical manifestations. Expanding testing to all pregnant women and clinics for sexually transmitted infections could help to unveil HTLV-1 asymptomatic carriers.This work was supported in part by grants from FIS-ISCIII PI-21/1717 and Fundación Mutua Madrileña AP-174112020

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2′-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design

Atopic dermatitis incidence and risk factors in young adults in Castellon (Spain): A prospective cohort study

Introduction: There are few atopic dermatitis (AD) incidence cohort studies in young adults, the etiology of this disease remains obscure, and AD risk factors in adults are not well understood. The objective of this study was to estimate AD ten-year incidence and prevalence in a cohort of adolescent aged 14–16 at inception in Castellon province in Valencia Region, Spain and describe related risk factors. Material and methods: From 2002 to 2012, a population-based prospective cohort study was carried out. Questionnaires from the International Study of Asthma and Allergies in Childhood (ISAAC) were used with an additional questionnaire for related factors completed by participants and their parents, respectively, in 2002. In 2012 the same questionnaires were completed by the participants’ through a telephone interview, and incidence and prevalence of AD were estimated. Directed acyclic graphs, Poisson regression and inverse probability weighted regression adjustment were used. Results: The participation rate was 79.5% (1435/1805) with AD lifetime prevalence of 34.9% and AD incidence of 13.5 per 1000 person years. Females presented higher prevalence and incidence than males. After adjustment significant risk factors were being female, history of asthma or allergic rhinitis, family history of AD, history of respiratory infections, history of bronchitis, history of pneumonia, history of sinusitis, and birthplace outside Castellon province. The highest AD population attributable risks were female, 30.3%, and history of respiratory infections 15.3%. Differences with AD childhood risk factors were found. Conclusions: AD incidence in our cohort was high and several risks factors were related to AD

Acute febrile illness is associated with Rickettsia spp infection in dogs

BACKGROUND: Rickettsia conorii is transmitted by Rhipicephalus sanguineus ticks and causes Mediterranean Spotted Fever (MSF) in humans. Although dogs are considered the natural host of the vector, the clinical and epidemiological significance of R. conorii infection in dogs remains unclear. The aim of this prospective study was to investigate whether Rickettsia infection causes febrile illness in dogs living in areas endemic for human MSF. METHODS: Dogs from southern Italy with acute fever (n = 99) were compared with case–control dogs with normal body temperatures (n = 72). Serology and real-time PCR were performed for Rickettsia spp., Ehrlichia canis, Anaplasma phagocytophilum/A. platys and Leishmania infantum. Conventional PCR was performed for Babesia spp. and Hepatozoon spp. Acute and convalescent antibodies to R. conorii, E. canis and A. phagocytophilum were determined. RESULTS: The seroprevalence rates at first visit for R. conorii, E. canis, A. phagocytophilum and L. infantum were 44.8%, 48.5%, 37.8% and 17.6%, respectively. The seroconversion rates for R. conorii, E. canis and A. phagocytophilum were 20.7%, 14.3% and 8.8%, respectively. The molecular positive rates at first visit for Rickettsia spp., E. canis, A. phagocytophilum, A. platys, L. infantum, Babesia spp. and Hepatozoon spp. were 1.8%, 4.1%, 0%, 2.3%, 11.1%, 2.3% and 0.6%, respectively. Positive PCR for E. canis (7%), Rickettsia spp. (3%), Babesia spp. (4.0%) and Hepatozoon spp. (1.0%) were found only in febrile dogs. The DNA sequences obtained from Rickettsia and Babesia PCRs positive samples were 100% identical to the R. conorii and Babesia vogeli sequences in GenBank®, respectively. Febrile illness was statistically associated with acute and convalescent positive R. conorii antibodies, seroconversion to R. conorii, E. canis positive PCR, and positivity to any tick pathogen PCRs. Fourteen febrile dogs (31.8%) were diagnosed with Rickettsia spp. infection based on seroconversion and/or PCR while only six afebrile dogs (12.5%) seroconverted (P = 0.0248). The most common clinical findings of dogs with Rickettsia infection diagnosed by seroconversion and/or PCR were fever, myalgia, lameness, elevation of C-reactive protein, thrombocytopenia and hypoalbuminemia. CONCLUSIONS: This study demonstrates acute febrile illness associated with Rickettsia infection in dogs living in endemic areas of human MSF based on seroconversion alone or in combination with PCR

Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting

Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2′-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3′-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2′-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold kcat/KM preference for ddT over thymidine, an overall 10 000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure–function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure–function relationship in dNKs

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing

Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing