136 research outputs found
Unraveling the Differences of the Hydrolytic Activity of Trypanosoma cruzi trans-Sialidase and Trypanosoma rangeli Sialidase: A Quantum MechanicsâMolecular Mechanics Modeling Study
Chagasâ disease, also known as American trypanosomiasis, is a lethal, chronic disease that currently affects more than 10 million people in Central and South America. The trans-sialidase from Trypanosoma cruzi (T. cruzi, TcTS) is a crucial enzyme for the survival of this parasite: sialic acids from the host are transferred to the cell surface glycoproteins of the trypanosome, thereby evading the hostâs immune system. On the other hand, the sialidase of T. rangeli (TrSA), which shares 70% sequence identity with TcTS, is a strict hydrolase and shows no trans-sialidase activity. Therefore, TcTS and TrSA represent an excellent framework to understand how different catalytic activities can be achieved with extremely similar structures. By means of combined quantum mechanicsâmolecular mechanics (QM/MM, SCC-DFTB/Amberff99SB) calculations and umbrella sampling simulations, we investigated the hydrolysis mechanisms of TcTS and TrSA and computed the free energy profiles of these reactions. The results, together with our previous computational investigations, are able to explain the catalytic mechanism of sialidases and describe how subtle differences in the active site make TrSA a strict hydrolase and TcTS a more efficient trans-sialidase.Fil: Bueren Calabuig, Juan A.. University of Florida; Estados UnidosFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Roitberg, AdriĂĄn. University of Florida; Estados Unido
Impact of Ser17 phosphorylation on the conformational dynamics of the oncoprotein MDM2
MDM2 is an important oncoprotein
that downregulates the activity
of the tumor suppressor protein p53 via binding of its N-terminal
domain to the p53 transactivation domain. The first 24 residues of
the MDM2 N-terminal domain form an intrinsically disordered âlidâ
region that interconverts on a millisecond time scale between âopenâ
and âclosedâ states in unliganded MDM2. While the former
conformational state is expected to facilitate p53 binding, the latter
competes in a pseudo-substrate manner with p53 for its binding site.
Phosphorylation of serine 17 in the MDM2 lid region is thought to
modulate the equilibrium between âopenâ and âclosedâ
lid states, but contradictory findings on the favored lid conformational
state upon phosphorylation have been reported. Here, the nature of
the conformational states of MDM2 pSer17 and Ser17Asp variants was
addressed by means of enhanced sampling molecular dynamics simulations.
Detailed analyses of the computed lid conformational ensembles indicate
that both lid variants stabilize a âclosedâ state, with
respect to wild type. Nevertheless, the nature of the closed-state
conformational ensembles differs significantly between the pSer17
and Ser17Asp variants. Thus, care should be applied in the interpretation
of biochemical experiments that use phosphomimetic variants to model
the effects of phosphorylation on the structure and dynamics of this
disordered protein region
From the Molecular Biology to the Gene Therapy of a DNA Repair Syndrome: Fanconi Anemia
Diseases & disorder
In vivo imaging of lung inflammation with neutrophil-specific Ga-68 nano-radiotracer
In vivo detection and quantification of inflammation is a major goal in molecular imaging. Furthermore, cell-specific detection of inflammation would be a tremendous advantage in the characterization of many diseases. Here, we show how this goal can be achieved through the synergistic combination of nanotechnology and nuclear imaging. One of the most remarkable features of this hybrid approach is the possibility to tailor the pharmacokinetics of the nanomaterial-incorporated biomolecule and radionuclide. A good example of this approach is the covalent binding of a large amount of a neutrophil-specific, hydrophobic peptide on the surface of Ga-68 core-doped nanoparticles. This new nano-radiotracer has been used for non-invasive in vivo detection of acute inflammation with very high in vivo labelling efficiency, i.e. a large percentage of labelled neutrophils. Furthermore, we demonstrate that the tracer is neutrophil-specific and yields images of neutrophil recruitment of unprecedented quality. Finally, the nano-radiotracer was successfully detected in chronic inflammation in atherosclerosis-prone ApoE(-/-) mice after several weeks on a high-fat diet
Terapias avanzadas en enfermedades raras
Advanced therapies are a group of medicines for human use based on gene therapy, somatic cell therapy or tissue-engineering. In this paper we present some general basic concepts, describe the most promising strategies and summarize the results of a variety of clinical trials for a significant group of monogenic inherited diseases. Major emphasis is placed on ex vivo gene therapy approaches for diseases of the hematopoietic system, since these corresponded to pioneering treatments that have guided research in this field. We also highlight some examples of successful in vivo gene therapy and point out the contribution of somatic cell therapy and tissue engineering to the group of rare skin diseases. We aim to provide an overview of the state of the art of advanced therapies in rare diseases and highlight some of the cutting-edge technologies under development that can provide more effective and safer treatments in the future.Las terapias avanzadas comprenden un grupo de medicamentos biolĂłgicos basados en la terapia gĂ©nica, la terapia celular y la ingenierĂa de tejidos. En este artĂculo se presentan algunos conceptos bĂĄsicos, se describen las estrategias mĂĄs prometedoras y se detallan los resultados de diferentes ensayos clĂnicos para un grupo significativo de enfermedades, especialmente las enfermedades hereditarias monogĂ©nicas. Se hace un mayor hincapiĂ© en la terapia gĂ©nica ex vivo en enfermedades del sistema hematopoyĂ©tico, al ser el grupo de enfermedades pioneras que ademĂĄs han orientado la investigaciĂłn en el campo. Destacamos tambiĂ©n algunos ejemplos de Ă©xito de terapia gĂ©nica in vivo y señalamos la contribuciĂłn de la terapia celular y de la ingenierĂa de tejidos al grupo de enfermedades raras de la piel. Pretendemos con ello dar una visiĂłn de la situaciĂłn de las terapias avanzadas en enfermedades raras y señalamos algunas de las vĂas de futuro orientadas al desarrollo de tratamientos mĂĄs eficaces y seguros
XPF-Dependent DNA Breaks and RNA Polymerase II Arrest Induced by Antitumor DNA Interstrand Crosslinking-Mimetic Alkaloids
SummaryTrabectedin and Zalypsis are two potent anticancer tetrahydroisoquinoline alkaloids that can form a covalent bond with the amino group of a guanine in selected triplets of DNA duplexes and eventually give rise to double-strand breaks. Using well-defined in vitro and in vivo assays, we show that the resulting DNA adducts stimulate, in a concentration-dependent manner, cleavage by the XPF/ERCC1 nuclease on the strand opposite to that bonded by the drug. They also inhibit RNA synthesis by: (1) preventing binding of transcription factors like Sp1 to DNA, and (2) arresting elongating RNA polymerase II at the same nucleotide position regardless of the strand they are located on. Structural models provide a rationale for these findings and highlight the similarity between this type of DNA modification and an interstrand crosslink
Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM
Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation
Epigenetic Alterations in Fanconi Anaemia : Role in Pathophysiology and Therapeutic Potential
Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3TÎČ and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population
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