The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible “hinge”. While this region has not been structurally resolved, it likely acts as an extended linker between the two cTnT functional domains. Mutations in this region cause phenotypically diverse and often severe forms of HCM. Mechanistic insight, however, has been limited by the lack of structural information. To overcome this limitation, we evaluated the effects of cTnT 160–163 mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models. R-IVM revealed that cTnT mutations Δ160E, E163R and E163K disrupted weak electrostatic actomyosin binding. Reducing the ionic strength or decreasing Brownian motion rescued function. This is the first observation of HCM-linked mutations in cTnT disrupting weak interactions between the thin filament and myosin. To evaluate the in vivo effects of altering weak actomyosin binding we generated transgenic mice expressing Δ160E and E163R mutant cTnT and observed severe cardiac remodeling and profound myofilament disarray. The functional changes observed in vitro may contribute to the structural impairment seen in vivo by destabilizing myofilament structure and acting as a constant pathophysiologic stress.
Mutations in the cardiac thin filament (TF) have highly variable effects on the regulatory function of the cardiac sarcomere. Understanding the molecular-level dysfunction elicited by TF mutations is crucial to elucidate cardiac disease mechanisms. The hypertrophic cardiomyopathy-causing cardiac troponin T (cTnT) mutation ⌬160Glu (⌬160E) is located in a putative "hinge" adjacent to an unstructured linker connecting domains TNT1 and TNT2. Currently, no high-resolution structure exists for this region, limiting significantly our ability to understand its role in myofilament activation and the molecular mechanism of mutation-induced dysfunction. Previous regulated in vitro motility data have indicated mutation-induced impairment of weak actomyosin interactions. We hypothesized that cTnT-⌬160E repositions the flexible linker, altering weak actomyosin electrostatic binding and acting as a biophysical trigger for impaired contractility and the observed remodeling. Using time-resolved FRET and an all-atom TF model, here we first defined the WT structure of the cTnT-linker region and then identified ⌬160E mutation-induced positional changes. Our results suggest that the WT linker runs alongside the C terminus of tropomyosin. The ⌬160E-induced structural changes moved the linker closer to the tropomyosin C terminus, an effect that was more pronounced in the presence of myosin subfragment (S1) heads, supporting previous findings. Our in silico model fully supported this result, indicating a mutation-induced decrease in linker flexibility. Our findings provide a framework for understanding basic pathogenic mechanisms that drive severe clinical hypertrophic cardiomyopathy phenotypes and for identifying structural targets for intervention that can be tested in silico and in vitro.
Genetic factors will determine the higher variability, which found in response to lipid reduction treatment (statins). However, due to ethnicity the frequency and effect of single nucleotide polymorphisms (SNPs) may differ. The main aim of lipid lowering medical treatment is to eventually prevent the endogenous production of cholesterol through inhibition of HMG-CoA reductase, The resulting decrease in hepatocyte cholesterol concentration triggers up-regulation of low-density lipoprotein (LDL)-receptor expression by inducing sterol regulatory element-binding protein (SREBP) 2 cleavage, as SREBP-2 activates LDL receptor transcription. The aim of this study was to evaluate the effects of low density lipoprotein receptor (LDL-R) gene variants (rs200727689; rs72658860) in response to atorvastatin treatment in atherosclerotic coronary artery disease (ACAD) in a sample of Iraqi patients. The genetic polymorphisms of rs200727689 and rs72658860 were studied in patients undergoing coronary angiography (CAG). One hundred Iraqi patients include 52 patients treated with 20mg/day and 48 patients treated with 40mg/day, In addition, 100 apparently healthy subjects were genotype for these SNP by the thermal profile of allelic discrimination methods used Real Time PCR (RT-PCR) assay. A significant increase in A allele frequency (rs200727689) compared with controls for total ACAD patients (43% vs 23.5%; OR= 2.46; 95% C.I: 1.60-3.77; p=0.000). For (rs72658860) SNP, among total patients the A allele also increased the frequency substantially compared with the controls (40.5% vs 23.5%; P=0.0003; OR= 2.22; 95% C.I: 1.44-3.41). In the patients who were treated with atorvastatin, SNP rs72658860 AA genotype significantly affected the response of atorvastatin in dose 20mg compared with 40mg. The result showed in AA genotype of (rs200727689) SNP a reduction in TC concentrations (277.55±108.38 vs 326.99±63.56) and LDL-C concentrations (198.33±100.0 vs 250.01±62.52) in sera compared with GG genotype in patients treated with 20 mg atorvastatin, although, none in all parameters statistically significant differences.
Systemic Lupus Erythematosus is a debilitating autoimmune disease that afflicts about 1.5 million Americans today. Although SLE is a multifactorial disease, SLE can arise with disruptions in two genes: Dnase1 and Dnase1L3. While the structure is known for DNase1, the structure for Dnase1L3 is unknown. To study the structure and function of the Dnase1L3 enzyme, we designed a heterologous expression system and purification protocol for mouse DNase1L3 in E. coli. This system will allow us to characterize the enzymatic activity of the wild-type enzyme. Towards the purpose of solving its 3D structure with X-Ray crystallography we proceeded to the crystallization step of Dnase1L3. A variety of culturing conditions, purification methods, and characterization techniques were performed on purified DNase1L3. After testing a few different methods, we successfully expressed DNase1-L3 in E. coli using Rosetta-gami cells as a fusion protein to Maltose Binding Protein. The fusion enzyme was isolated via amylose affinity resin. After elution from the affinity column, the enzyme was cleaved with TEV protease and further purified on S-resin as a defined peak. Finally, Dnase1L3 was purified using size exclusion chromatography. The activity of purified DNase1-L3 was assessed through digestion of plasmid DNA. The identify of Dnase1L3 was confirmed by Western blot. Now that we have obtained an efficient purification method, verified the activity, and confirmed identity with specific antibodies, we are interrogating the structural aspects of the enzyme through X-Ray crystallography. The structural composition of this enzyme will be invaluable as pertaining to possible use in treating Systemic Lupus Erythematosus.
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