The N-Terminal domain of SIRT1 is a positive regulator of endogenous SIRT1-dependent deacetylation and transcriptional outputs

SummaryThe NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating many different proteins, including histones and transcription factors. The mechanisms controlling SIRT1 enzymatic activity are complex and incompletely characterized, yet essential for understanding how to develop therapeutics that target SIRT1. Here, we demonstrate that the N-terminal domain of SIRT1 (NTERM) can trans-activate deacetylation activity by physically interacting with endogenous SIRT1 and promoting its association with the deacetylation substrate NF-κB p65. Two motifs within the NTERM domain contribute to activation of SIRT1-dependent activities, and expression of one of these motifs in mice is sufficient to lower fasting glucose levels and improve glucose tolerance in a manner similar to overexpression of SIRT1. Our results provide insights into the regulation of SIRT1 activity and a rationale for pharmacological control of SIRT1-dependent activities

The Structure of the NPC1L1 N-Terminal Domain in a Closed Conformation

NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket

Sterol Binding and Specificity of NPC1L1(NTD).

<p>(A) Cholesterol binding. Each reaction, in a final volume of 100 µl, contained 0.5 pmol of purified HIS6-NPC1L1(NTD) and the indicated concentration of ³H-cholesterol in the absence (•) or presence (○) of 100 uM unlabeled cholesterol. Bound ³H-cholesterol was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018722#s4" target="_blank"><i>Materials and Methods</i></a>. Each data point represents the average of triplicate assays. (B) Competitive binding of ³H-cholesterol in the presence of unlabeled sterols. Each reaction, in a total volume of 100 µl, contained 0.5 pmol HIS6-NPC1L1(NTD), 10 nM ³H-cholesterol and 1 µM of the indicated unlabeled sterol. Each data point represents the average of triplicate assays and represents the amount of ³H-cholesterol bound relative to that in the control tube, which did not contain unlabeled sterol. (C) Competitive binding of ³H-cholesterol in the presence of unlabeled sterols. Each reaction, in a total volume of 100 µl, contained 0.5 pmol HIS6-NPC1L1(NTD), 10 nM ³H-cholesterol and varying concentrations of the indicated unlabeled sterol. Each data point represents the average of triplicate assays and represents the amount of ³H-cholesterol bound relative to that in the control tube, which did not contain unlabeled sterol.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

NPC1L1(NTD) in a closed conformation.

<p>(A) The surface of NPC1L1(NTD), colored green, reveals a cholesterol binding pocket (blue) that is closed to solvent. (B) The surface of NPC1(NTD), colored gray, shows the cholesterol binding pocket (yellow) exposed to solvent. The isooctyl side chain of cholesterol (stick model in green) is visible and solvent exposed. (C) Cutaway view of the cholesterol binding pocket of NPC1L1(NTD) in the closed conformation. (D) Cutaway view of the cholesterol binding pocket of NPC1(NTD) in an open conformation.</p

The Structure of NPC1L1(NTD).

<p>(A) NPC1L1(NTD) is represented as a ribbon diagram and the disulfide bonds are shown in yellow. Domain A is colored orange (α-helices), cyan (β-sheets), and gray (loops). α3 (red), α7 (blue), and the α8/β7 loop (green) surround the entrance of the cholesterol binding pocket. Domain B is colored magenta. (B) Superposition of NPC1L1(NTD) (yellow) and NPC1(NTD) (gray). Bound cholesterol in NPC1 is shown as a stick model. Regions around the entrance of the cholesterol binding pocket in NPC1L1 are colored red (α3), blue (α7), and green (α8/β7 loop). (C) Sequence alignment of NPC1L1(NTD) and NPC1(NTD). N-linked glycosylation sites are shaded gray. Residues lining the cholesterol binding pocket are shaded yellow in NPC1. In NPC1L1, residues within the interior of the closed cholesterol binding pocket are shaded blue, residues on the exterior of the closed cholesterol binding pocket are shaded green, and residues separating the interior from the exterior are shaded red. Regions around the entrance to the cholesterol binding pocket that change conformation are boxed. The secondary structure of NPC1L1(NTD) is shown below the sequence.</p

NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann–Pick C1 (NPC1) and soluble Niemann–Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25–264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [3H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants

Competitive binding of ³H-LDV in the presence of unlabeled inhibitor.

<p>Each reaction, in a final volume of 200 μl, contained 50 nM NS5A-6HIS, 30 nM ³H-LDV and the indicated concentration of unlabeled LDV (●) or DCV (). % Bound represents the amount of ³H-LDV bound relative to that in the control tube, which contained no unlabeled inhibitor. Each data point represents the average of at least 3 assays. K_i was calculated using the Cheng-Prusoff equation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122844#pone.0122844.ref030" target="_blank">30</a>]. EC₅₀ represents the 50% effective inhibitory concentration of HCV RNA replication in the Renilla luciferase genotype 1b, Con 1 replicon cell line.</p