Structural characterization of the histone variant macroH2A

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

Structure of the Dengue virus helicase/nucleoside triphosphatase catalytic domain at a resolution of 2.4 A

Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 A resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3' single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.     

Stearoyl-CoA desaturase 1 deficiency increases CTP:choline cytidylyltransferase translocation into the membrane and enhances phosphatidylcholine synthesis in liver

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in monounsaturated fatty acid synthesis. Previously, we showed that Scd1 deficiency reduces liver triglyceride accumulation and considerably decreases synthesis of very low density lipoprotein and its secretion in both lean and obese mice. In the present study, we found that Scd1 deficiency significantly modulates hepatic glycerophospholipid profile. The content of phosphatidylcholine (PC) was increased by 40% and the activities of CTP:choline cytidylyltransferase (CCT), the rate-limiting enzyme in de novo PC synthesis, and choline phosphotransferase were increased by 64 and 53%, respectively, in liver of Scd1-/- mice. In contrast, the protein level of phosphatidylethanolamine N-methyltransferase, an enzyme involved in PC synthesis via methylation of phosphatidylethanolamine, was decreased by 80% in the liver of Scd1-/- mice. Membrane translocation of CCT is required for its activation. Immunoblot analyses demonstrated that twice as much CCTalpha was associated with plasma membrane in livers of Scd1-/- compared with wild type mice, suggesting that Scd1 mutation leads to an increase in CCT membrane affinity. The incorporation of [(3)H]glycerol into PC was increased by 2.5-fold in Scd1-/- primary hepatocytes compared with those of wild type mice. Furthermore, mitochondrial glycerol-3-phosphate acyltransferase activity was reduced by 42% in liver of Scd1-/- mice; however, the activities of microsomal glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, and ethanolamine phosphotransferase were not affected by Scd1 mutation. Our study revealed that SCD1 deficiency specifically increases CCT activity by promoting its translocation into membrane and enhances PC biosynthesis in liver.     

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Induction of brain natriuretic peptide and monocyte chemotactic protein-1 gene expression by oxidized low-density lipoprotein: relevance to ischemic heart failure

Brain natriuretic peptide (BNP) and monocyte chemotactic protein-1 (MCP-1) are biomarkers of heart failure (HF). The aim of the present study was to determine the role of oxidized low-density lipoprotein (Ox-LDL) in the induction of these biomarkers and the signaling pathways involved in vitro. Incubation of HL-1 cardiomyocytes and human myocytes with Ox-LDL induced the expression of BNP and MCP-1 genes, while native LDL had no effect. When peroxides associated with Ox-LDL were reduced to hydroxides, the ability to induce BNP and MCP-1 gene expression was abolished. Furthermore, exposure of HL-1 cells to ischemic conditions alone had no effect on BNP gene expression, while ischemia followed by reperfusion resulted in increased expression of BNP gene. Inhibitors of ERK and JNK inhibited the induction of BNP. Signaling array results suggested that the induction of both MAPK and NF-κB pathways is involved in the induction of BNP by Ox-LDL. These results suggest that Ox-LDL or peroxidized lipids formed in oxidatively stressed myocytes during ischemia-reperfusion injury may play a role in the induction of BNP and MCP-1.     

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.