Lydia Blachowicz

Effect of Immune Deficiency on Lipoproteins and Atherosclerosis in Male Apolipoprotein E–Deficient Mice

Abstract—To determine whether T cells and B cells influence lipid metabolism and atherosclerosis, we crossed apolipoprotein E-deficient (apoE°) mice with recombination activating gene 2-deficient (RAG2°) mice. Total plasma cholesterol levels were ≈20% higher in male apoE° mice compared with the apoE°RAG2° mice at 8 weeks of age, and plasma triglyceride levels were 2.5-fold higher in the apoE° mice even when plasma cholesterol levels were similar. Male mice with plasma cholesterol levels between 400 and 600 mg/dL at 8 weeks of age were euthanized at 27 and 40 weeks of age. The aortic root lesion area in the apoE°RAG2° mice, compared with that in the immune-competent apoE° mice, was 81% and 57% smaller at 27 and 40 weeks of age, respectively. In contrast, there was no difference in the size of the brachiocephalic trunk lesions. Similar results were obtained with mice euthanized at 40 weeks of age that had 8-week cholesterol levels between 300 and 399 mg/dL. In apoE°RAG2° mice, aortic root atherosclerosis was more profoundly suppressed at lower cholesterol levels. Thus, T and B cells and their products differentially influence the development of atherosclerosis at different sites. We also demonstrate a profound effect of the immune system on plasma lipid homeostasis.

Multidomain assembled states of Hck tyrosine kinase in solution

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.

Genetic Background Selectively Influences Innominate Artery Atherosclerosis: Immune System Deficiency as a Probe

Objective—We sought to examine whether there is a site-specific effect on atherosclerosis of the absence of mature T and B cells caused by a recombination activating-gene deficiency in LDL receptor-deficient mice and whether this effect is influence by the extent of backcrossing to C57BL/6 mice. Methods and Results—Male mice were fed atherogenic diets for 3 months. In strain 1 mice, in which ≈93% of the genes were from C57BL/6 mice, the absence of mature T and B cells led to a significant reduction in atherosclerosis in both the aortic sinus and the innominate artery. In strain 2 mice, in which ≈99+% of the genes were from C57BL/6 mice, immune system deficiency led to a site-specific effect on atherosclerosis, with a reduction in atherosclerosis in the aortic sinus but not in the innominate artery, similar to previous results obtained with apolipoprotein E−/− mice. All of the immune system-incompetent mice had lower plasma total and VLDL cholesterol levels regardless of strain or diet, indicating that differences in lipid levels were unlikely to be responsible for these site-specific effects of immune system deficiency. Conclusions—These results suggest that immune system deficiency has a site-specific effect on atherosclerosis that is sensitive to the genetic background of the mice.

A Conformational Intermediate in Glutamate Receptor Activation

Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ∼30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure

In voltage-gated K+ channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn2+ or Cd2+ with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by ∼7–8 Å and rotated counterclockwise by 37° along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

A structural study of ion permeation in OmpF porin from anomalous X-ray diffraction and molecular dynamics simulations.

OmpF, a multiionic porin from Escherichia coli, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br(–) ions from four different crystal structures and compared with Mg(2+) sites and Rb(+) sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin.

Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

1H, 15N, and 13C resonance assignments of the intrinsically disordered SH4 and Unique domains of Hck

Hematopoietic cell kinase (Hck) is an important signaling enzyme and a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. The protein shares the same SH4–Unique–SH3–SH2–kinase multi-domain architecture as the other eight members of the Src family of non-receptor tyrosine kinases. These enzymes are often found anchored to the intracellular side of the membrane via lipidation of the SH4 domain and are integral components of signaling cascades localized at the cell surface. Despite the detailed structural information available for the SH3, SH2, and kinase domains of Hck, the intrinsically disordered nature of the SH4 and Unique domains has resulted in a lack of information for this important region of the protein that is responsible for membrane association. Here, we report the 1H, 15N and 13C chemical shifts of the Hck SH4–Unique domains at pH 4.5.

Structural and Functional Characterization of a Calcium-Activated Cation Channel From Tsukamurella Paurometabola

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a preliminary characterization of this calcium-gated cation channel.