A Comparative Molecular Dynamics Study of Methylation State Specificity of JMJD2A

Histone modifications have great importance in epigenetic regulation. JMJD2A is a histone demethylase which is selective for di- and trimethyl forms of residues Lys9 and Lys36 of Histone 3 tail (H3K9 and H3K36). We present a molecular dynamics simulations of mono-, di- and trimethylated histone tails in complex with JMJD2A catalytic domain to gain insight into how JMJD2A discriminates between the methylation states of H3K9. The methyl groups are located at specific distances and orientations with respect to Fe(II) in methylammonium binding pocket. For the trimethyllysine the mechanism which provides the effectual orientation of methyl groups is the symmetry, whereas for the dimethyllysine case the determining factors are the interactions between methyllysine head and its environment and subsequently the restriction on angular motion. The occurrence frequency of methyl groups in a certain proximity of Fe(II) comes out as the explanation of the enzyme activity difference on di- and tri-methylated peptides. Energy analysis suggests that recognition is mostly driven by van der Waals and followed by Coulombic interactions in the enzyme-substrate interface. The number (mono, di or tri) and orientations of methyl groups and water molecules significantly affect the extent of van der Waals interaction strengths. Hydrogen bonding analysis suggests that the interaction between JMJD2A and its substrates mainly comes from main chain-side chain interactions. Binding free energy analysis points out Arg8 as an important residue forming an intra-substrate hydrogen bond with tri and dimethylated Lys9 of the H3 chain. Our study provides new insights into how JMJD2A discriminates between its substrates from both a structural and dynamical point of view

Neuropathology of 16p13.11 Deletion in Epilepsy

16p13.11 genomic copy number variants are implicated in several neuropsychiatric disorders, such as schizophrenia, autism, mental retardation, ADHD and epilepsy. The mechanisms leading to the diverse clinical manifestations of deletions and duplications at this locus are unknown. Most studies favour NDE1 as the leading disease-causing candidate gene at 16p13.11. In epilepsy at least, the deletion does not appear to unmask recessive-acting mutations in NDE1, with haploinsufficiency and genetic modifiers being prime candidate disease mechanisms. NDE1 encodes a protein critical to cell positioning during cortical development. As a first step, it is important to determine whether 16p13.11 copy number change translates to detectable brain structural alteration. We undertook detailed neuropathology on surgically resected brain tissue of two patients with intractable mesial temporal lobe epilepsy (MTLE), who had the same heterozygous NDE1-containing 800 kb 16p13.11 deletion, using routine histological stains and immunohistochemical markers against a range of layer-specific, white matter, neural precursor and migratory cell proteins, and NDE1 itself. Surgical temporal lobectomy samples from a MTLE case known not to have a deletion in NDE1 and three non-epilepsy cases were included as disease controls. We found that apart from a 3 mm hamartia in the temporal cortex of one MTLE case with NDE1 deletion and known hippocampal sclerosis in the other case, cortical lamination and cytoarchitecture were normal, with no differences between cases with deletion and disease controls. How 16p13.11 copy changes lead to a variety of brain diseases remains unclear, but at least in epilepsy, it would not seem to be through structural abnormality or dyslamination as judged by microscopy or immunohistochemistry. The need to integrate additional data with genetic findings to determine their significance will become more pressing as genetic technologies generate increasingly rich datasets. Detailed examination of brain tissue, where available, will be an important part of this process in neurogenetic disease specifically

Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands

Structural Characterization of a Novel Chlamydia pneumoniae Type III Secretion-Associated Protein, Cpn0803

Type III secretion (T3S) is an essential virulence factor used by Gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S

To automate or not to automate: this is the question

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected

Human macrophages differentiated in the presence of vitamin D3 restrict dengue virus infection and innate responses by downregulating mannose receptor expression

ABSTARCT: Severe dengue disease is associated with high viral loads and overproduction of pro-inflammatory cytokines, suggesting impairment in the control of dengue virus (DENV) and the mechanisms that regulate cytokine production. Vitamin D3 has been described as an important modulator of immune responses to several pathogens. Interestingly, increasing evidence has associated vitamin D with decreased DENV infection and early disease recovery, yet the molecular mechanisms whereby vitamin D reduces DENV infection are not well understood. METHODS AND PRINCIPAL FINDINGS: Macrophages represent important cell targets for DENV replication and consequently, they are key drivers of dengue disease. In this study we evaluated the effect of vitamin D3 on the differentiation of monocyte-derived macrophages (MDM) and their susceptibility and cytokine response to DENV. Our data demonstrate that MDM differentiated in the presence of vitamin D3 (D3-MDM) restrict DENV infection and moderate the classical inflammatory cytokine response. Mechanistically, vitamin D3-driven differentiation led to reduced surface expression of C-type lectins including the mannose receptor (MR, CD206) that is known to act as primary receptor for DENV attachment on macrophages and to trigger of immune signaling. Consequently, DENV bound less efficiently to vitamin D3-differentiated macrophages, leading to lower infection. Interestingly, IL-4 enhanced infection was reduced in D3-MDM by restriction of MR expression. Moreover, we detected moderate secretion of TNF-α, IL-1β, and IL-10 in D3-MDM, likely due to less MR engagement during DENV infection. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a molecular mechanism by which vitamin D counteracts DENV infection and progression of severe disease, and indicates its potential relevance as a preventive or therapeutic candidate

Nudel and FAK as Antagonizing Strength Modulators of Nascent Adhesions through Paxillin

Competition for binding to the cellular protein paxillin by the proteins Nudel and focal adhesion kinase is important for the proper regulation of cell adhesion and migration