Isoform- and cell-state-specific lipidation of ApoE in astrocytes.

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP-binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10-20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.

The HPK1 Inhibitor A-745 Verifies the Potential of Modulating T Cell Kinase Signaling for Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

In vitro and in cellulo ApoE particle formation, isolation, and characterization.

Apolipoprotein E (ApoE) particles are responsible for packing and transporting lipids throughout aqueous environments. We detail steps to assess in vitro particles forming from artificial membranes using right-angle light scattering and to measure their size using dynamic light scattering. We further describe how to generate in cellulo ApoE particles containing triacylglycerol under fatty-acid-induced stress. We also detail steps to isolate them from cell secretome by immunoprecipitation and analyze their lipid cargo by thin-layer chromatography. For complete details on the use and execution of this protocol, please refer to Lindner et al. (2022).1.

Fragment-Based Discovery of an Apolipoprotein E4 (apoE4) Stabilizer.

Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

SAR and characterization of non-substrate isoindoline urea inhibitors of nicotinamide phosphoribosyltransferase (NAMPT).

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors.

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.

Quantification of TRPV1 protein levels in rat tissues to understand its physiological roles.

Transient receptor potential subfamily V, member 1 (TRPV1) is a nonselective cation channel expressed in both the peripheral and central nervous systems (CNS). TRPV1 protein levels in rat tissues were determined under normal and pain states using enzyme-linked immunosorbent assay. In naive rats, brain TRPV1 protein concentrations ranged from 1.5 to 4 ng/mg in hippocampus, cortex, hypothalamus, and cerebellum. Rat spinal cord TRPV1 protein levels were 40-50 ng/mg in L1-L5 of the lumbar regions, but increased to 97 ± 9.3 ng/mg toward the end of the lumbar region (L6-S1). In the complete Freund's adjuvant (CFA)-induced inflammatory pain model, TRPV1 protein level significantly increased on both the contralateral (36.5 %, p < 0.05) and ipsilateral (31.4 %, p < 0.05) L4-L6 dorsal root ganglia (DRG). TRPV1 protein levels also increased 33.3 % (p < 0.05) on the ipsilateral sciatic nerve, but no significant change in the lumbar spinal cord of CFA rats. In the monoiodoacetate-induced rat knee joint pain model, TRPV1 protein level was significantly reduced in the ipsilateral L3-L5 DRG (33.3 %, p < 0.01), no significant difference was detected in the lumbar region of the spinal cord. Quantitative determination of TRPV1 protein levels may help to elucidate the TRPV1 physiological roles and regulatory mechanisms in various pain states.

HPLC-SEC characterization of membrane protein-detergent complexes.

Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes.

Development of ELISA to measure TRPV1 protein in rat tissues.

The transient receptor potential vanilloid receptor type 1 (TRPV1) is a non-selective cation channel expressed in both the peripheral and the central nervous systems. To quantitatively determine TRPV1 protein levels in native rat tissues, novel monoclonal antibodies were raised against full-length recombinant human TRPV1 protein and utilized to develop a sandwich ELISA assay. Monoclonal antibody 10E3-1A2 specifically recognized TRPV1 protein and the recognition epitope was determined to reside in amino acids 45-58 of human and rat TRPV1. Using the TRPV1 polyclonal antibody ABRK4 as the capturing antibody and the monoclonal antibody 10E3-1A2 as the detection antibody, a sandwich ELISA that detected both human and rat TRPV1 protein was established. Recombinant human TRPV1 heterologously expressed in mammalian HEK293-F cells, which showed high ligand-binding affinity, was purified by TRPV1 monoclonal antibody affinity chromatography and used as protein standard to quantify TRPV1 protein levels. This ELISA detected TRPV1 protein as low as 1.5ng/ml (15pM), and was able to determine TRPV1 protein levels in native rat tissues such as DRG and spinal cord. This is the first TRPV1 sandwich ELISA that determines the abundance of TRPV1 protein in different tissues. It provides a powerful tool to quantify changes of TRPV1 protein levels in pathological states.

Expression and purification of human TRPV1 in baculovirus-infected insect cells for structural studies.

TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.

Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli.

Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.

Glutamine 53 is a gatekeeper residue in the FK506 binding protein.

The effect of non-random conformational averaging in the urea-unfolded state on the folding pathway has been investigated in a variant of the FK506 binding protein with three additional residues at the amino terminus (FKBP(*)). Three mutations (asparagine, aspartate, and threonine) were introduced into position Q53 to enhance formation of non-native helix observed in this part of the protein in the urea-unfolded state. NMR analysis showed minor structural changes in the native state of each mutant, but additional medium-range alphaN(i,i+2) of each mutant nuclear Overhauser enhancements were observed in the urea-unfolded state that were not in FKBP(*), indicating that the mutations had a more substantial effect on the unfolded state ensemble than on the native state ensemble. Isothermal equilibrium denaturation measurements showed that the Q53T and Q53D mutants were destabilized, whereas the Q53N mutant was stabilized relative to FKBP(*) with little change in the equilibrium m values. The unfolding rates of Q53N and Q53T were similar to that of FKBP(*), but Q53D unfolded twice as fast as FKBP(*). In contrast, the mutations had a more pronounced effect on the refolding kinetics. Q53N refolded slightly faster and exhibited a kinetic folding intermediate similar to that of FKBP(*). The Q53D and Q53T mutants also refolded faster than FKBP(*) but lacked the folding intermediate, indicating that these mutants experienced a different folding trajectory and transition state than FKBP(*) and Q53N. The refolding kinetic Phi values were 0.74, 1.4 and 7.9 for Q53N, Q53T, and Q53D, respectively. The data point to Q53 functioning as a gatekeeper residue in the folding of FKBP(*). This study shows that perturbing the unfolded state ensemble via mutagenesis can provide insights into residues that play important roles in the folding pathway, and represents an attractive strategy for mapping the high-energy portions of the folding energy landscape.