Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

Angiopoietin-like protein 4/8 complex-mediated plasmin generation leads to cleavage of the complex and restoration of LPL activity.

Triglyceride (TG) metabolism is highly regulated by angiopoietin-like protein (ANGPTL) family members [Y. Q. Chen et al., J. Lipid Res. 61, 1203-1220 (2020)]. During feeding, ANGPTL8 forms complexes with the fibrinogen-like domain-containing protein ANGPTL4 in adipose tissue to decrease ANGPTL3/8- and ANGPTL4-mediated lipoprotein lipase (LPL)-inhibitory activity and promote TG hydrolysis and fatty acid (FA) uptake. The ANGPTL4/8 complex, however, tightly binds LPL and partially inhibits it in vitro. To try to reconcile the in vivo and in vitro data on ANGPTL4/8, we aimed to find novel binding partners of ANGPTL4/8. To that end, we performed pulldown experiments and found that ANGPTL4/8 bound both tissue plasminogen activator (tPA) and plasminogen, the precursor of the fibrinolytic enzyme plasmin. Remarkably, ANGPTL4/8 enhanced tPA activation of plasminogen to generate plasmin in a manner like that observed with fibrin, while minimal plasmin generation was observed with ANGPTL4 alone. The addition of tPA and plasminogen to LPL-bound ANGPTL4/8 caused rapid, complete ANGPTL4/8 cleavage and increased LPL activity. Restoration of LPL activity in the presence of ANGPTL4/8 was also achieved with plasmin but was blocked when catalytically inactive plasminogen (S760A) was added to tPA or when plasminogen activator inhibitor-1 was added to tPA + plasminogen, indicating that conversion of plasminogen to plasmin was essential. Together, these results suggest that LPL-bound ANGPTL4/8 mimics fibrin to recruit tPA and plasminogen to generate plasmin, which then cleaves ANGPTL4/8, enabling LPL activity to be increased. Our observations thus reveal a unique link between the ANGPTL4/8 complex and plasmin generation.

Inverse association between apolipoprotein C-II and cardiovascular mortality: role of lipoprotein lipase activity modulation.

AIMS: Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II-mediated LPL activation is unclear. METHODS AND RESULTS: ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7-10.7) years. Apolipoprotein C-II-mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)-LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1-LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1-LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. CONCLUSION: The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1-LPL enzymatic activity.

Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

After feeding, adipose tissue lipoprotein lipase (LPL) activity should be maximized, therefore the potent LPL-inhibitory activity of angiopoietin-like protein 4 (ANGPTL4) must be blocked by ANGPTL8 through formation of ANGPTL4/8 complexes. ANGPTL4/8 tightly binds and protects LPL but also partially inhibits its activity. Recently, we demonstrated ANGPTL4/8 also binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin that cleaves ANGPTL4/8 to restore LPL activity. Although fully active LPL in the fat postprandially is desirable, ANGPTL4/8 removal could subject LPL to profound inhibition by ANGPTL3/8 (the most potent circulating LPL inhibitor), inhibition by other LPL inhibitors like ANGPTL4, ANGPTL3, and ApoC3 or interfere with ApoC2-mediated LPL activation. To understand better these potential paradoxes, we examined LPL inhibition by ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 and LPL stimulation by ApoC2 in the presence of ANGPTL4/8 + tPA + plasminogen. Remarkably, ANGPTL3/8-mediated LPL inhibition was almost completely blocked, with the mechanism being cleavage of fibrinogen-like domain-containing ANGPTL3 present in the ANGPTL3/8 complex. The LPL-inhibitory effects of ANGPTL4, ANGPTL3, and ApoC3 were also largely reduced in the presence of ANGPTL4/8 + tPA + plasminogen. In contrast, the ability of ApoC2 to stimulate LPL activity was unaffected by ANGPTL4/8-mediated plasmin generation. Together, these results explain how plasmin generated by increased postprandial ANGPTL4/8 levels in adipose tissue enables maximal LPL activity by preventing ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 from inhibiting LPL, while permitting ApoC2-mediated LPL activation to occur.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

ApoA5 lowers triglyceride levels via suppression of ANGPTL3/8-mediated LPL inhibition.

Triglyceride (TG) molecules represent the major storage form of fatty acids, and TG metabolism is essential to human health. However, the mechanistic details surrounding TG metabolism are complex and incompletely elucidated. Although it is known that angiopoietin-like protein 8 (ANGPTL8) increases TGs through an ANGPTL3/8 complex that inhibits LPL, the mechanism governing ApoA5, which lowers TGs, has remained elusive. Current hypotheses for how ApoA5 acts include direct stimulation of LPL, facilitation of TG-containing particle uptake, and regulation of hepatic TG secretion. Using immunoprecipitation-MS and Western blotting, biolayer interferometry, functional LPL enzymatic assays, and kinetic analyses of LPL activity, we show that ApoA5 associates with ANGPTL3/8 in human serum and most likely decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition. We also demonstrate that ApoA5 has no direct effect on LPL, nor does it suppress the LPL-inhibitory activities of ANGPTL3, ANGPTL4, or ANGPTL4/8. Importantly, ApoA5 suppression of ANGPTL3/8-mediated LPL inhibition occurred at a molar ratio consistent with the circulating concentrations of ApoA5 and ANGPTL3/8. Because liver X receptor (LXR) agonists decrease ApoA5 expression and cause hypertriglyceridemia, we investigated the effect of the prototypical LXR agonist T0901317 on human primary hepatocytes. We observed that T0901317 modestly stimulated hepatocyte ApoA5 release, but markedly stimulated ANGPTL3/8 secretion. Interestingly, the addition of insulin to T0901317 attenuated ApoA5 secretion, but further increased ANGPTL3/8 secretion. Together, these results reveal a novel intersection of ApoA5 and ANGPTL3/8 in the regulation of TG metabolism and provide a possible explanation for LXR agonist-induced hypertriglyceridemia.

Angiopoietin-like protein 4(E40K) and ANGPTL4/8 complex have reduced, temperature-dependent LPL-inhibitory activity compared to ANGPTL4.

We previously demonstrated that angiopoietin-like 8 (ANGPTL8) forms a localized complex with ANGPTL4 to reduce its lipoprotein lipase (LPL)-inhibitory activity and enable increased postprandial uptake of fatty acids (FA) into adipose tissue. Because prolonged cold exposure may increase adipose tissue FA uptake and decrease circulating triglycerides (TG) by reducing ANGPTL4 expression and inducing ANGPTL8 expression (and thus ANGPTL4/8 expression), we investigated the effect of temperature on ANGPTL4 and ANGPTL4/8 LPL-inhibitory activities in vitro. As the ANGPTL4(E40K) mutation results in decreased TG, we also characterized ANGPTL4(E40K) and ANGPTL4(E40K)/8 complex LPL-inhibitory activities. Interestingly, while ANGPTL3, ANGPTL3/8, and ANGPTL4 showed similar LPL inhibition at 37 °C and 22 °C, the already reduced LPL-inhibitory activity of ANGPTL4/8 at 37 °C was even more decreased at 22 °C. At 37 °C, ANGPTL4(E40K) manifested decreased LPL-inhibitory activity compared to ANGPTL4/8, while ANGPTL4(E40K)/8 had even further reduced potency. Remarkably, ANGPTL4/8, ANGPTL4(E40K), and ANGPTL4(E40K)/8 were each actually capable of stimulating LPL activity at 22 °C. Together, these results indicate that ANGPTL4/8 stimulation of LPL activity at low temperatures may represent an additional mechanism for further increasing adipose tissue FA uptake during cold exposure, beyond that already occurring due to decreased ANGPTL4 expression and increased ANGPTL8 expression. In addition, because ANGPTL4(E40K) has decreased LPL-inhibitory activity compared to ANGPTL4/8, our findings also suggest why ANGPTL4(E40K) carriers have decreased circulating TG levels.

Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids.

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Modulation of TARP γ8-Containing AMPA Receptors as a Novel Therapeutic Approach for Chronic Pain.

Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

Crystal structure of the human PRMT5:MEP50 complex.

Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Gain and loss of function of P2X7 receptors: mechanisms, pharmacology and relevance to diabetic neuropathic pain.

BACKGROUND: Genetic causes of exaggerated or reduced pain sensitivity in humans are well known. Recently, single nucleotide polymorphisms (SNPs) in the gene P2RX7, coding for the ATP-gated ion channel P2X7, have been described that cause gain-of-function (GOF) and loss-of-function (LOF), respectively of this channel. Importantly, P2RX7 SNPs have been associated with more or less severe pain scores in patient suffering of post-mastectomy pain and osteoarthritis. RESULTS: The functional consequences of some P2RX7 SNPs (rs208294 (His155Tyr), rs1718119 (Ala348Thr) and rs3751143 (Glu496Ala)) were studied in recombinant cells in vitro. Our findings suggest a correlation between GOF and LOF of P2X7 and actual channel protein expression. Both channel and pore function for these mutant P2X7 receptors changed in parallel to protein levels. On the other hand, the mutant receptors did not differ in their sensitivity to known P2X7 agonists and antagonists. We further demonstrated that in patients with diabetic peripheral neuropathic pain (DPNP), the presence of the GOF SNPs rs208294 (His155Tyr) and rs1718119 (Ala348Thr) is associated, in females, with higher pain intensity scores. CONCLUSIONS: Our present results confirm the physiological relevance of some of the SNPs in the P2RX7 gene and show that the presence of these genetic variants correlates with pain sensitivity also in a diabetic neuropathic pain patient population.

Pharmacogenomics of gemcitabine metabolism: functional analysis of genetic variants in cytidine deaminase and deoxycytidine kinase.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

Development of a quantitative biochemical and cellular sphingomyelin synthase assay using mass spectrometry.

The last step in sphingolipid biosynthesis is the conversion of ceramide (Cer) to sphingomyelin (SM), which is catalyzed by sphingomyelin synthase (SMS). Two isoforms of SMS have been identified with differential subcellular localizations. It is not clear whether the two isoforms have any differences in biochemical or cellular SMS activities. This report describes a mass spectrometry (MS)-based method that was used to characterize biochemical and cellular SMS activities of the two isoforms of SMS, namely SMS1 and SMS2. Cellular extracts of SMS1 or SMS2 expressed in SF9 cells displayed significant SMS activity. When these activities were measured by MS, both SMS1 and SMS2 demonstrated similar time- and substrate-dependent SMS activity. A previously reported SMS inhibitor, D609, inhibited both SMS1 and SMS2 activity. In HEK293 cells, overexpression of either SMS1 or SMS2 significantly increased SMS activity. These studies using MS methods to measure SMS activity of SMS1 and SMS2 represent the first quantitative measurement of SMS activities. The establishment of quantitative biochemical and cellular SMS assays may help to facilitate the discovery of novel SMS1- or SMS2-specific inhibitors.

A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

A sensitive and drug tolerant assay for detecting anti-AAV9 antibodies using affinity capture elution.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

Structure-Based Design of Active-Site-Directed, Highly Potent, Selective, and Orally Bioavailable Low-Molecular-Weight Protein Tyrosine Phosphatase Inhibitors.

Protein tyrosine phosphatases constitute an important class of drug targets whose potential has been limited by the paucity of drug-like small-molecule inhibitors. We recently described a class of active-site-directed, moderately selective, and potent inhibitors of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Here, we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (Ki = 1.2 nM, >8000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak cytochrome P450 inhibition, high metabolic stability, moderate to high cell permeability (Papp > 0.2 nm/s), and moderate to good oral bioavailability (% F from 23 to 50% in mice), and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.

Role of ubiquitination in PCSK9-mediated low-density lipoprotein receptor degradation.

The proprotein convertases subtilisin kexin 9 (PCSK9) binds to the epidermal growth factor domain A (EGF-A) of low-density lipoprotein receptor (LDLR) and leads to its destruction. However, the intracellular processes leading to LDLR degradation have not been fully delineated. In this report, we show that PCSK9 treatment can lead to ubiquitination of LDLR, which was enhanced in the presence of proteasome inhibitor MG132. Furthermore, LDLR protein carrying mutations in the C-terminal ubiquitination sites was resistant to PCSK9-mediated degradation. Our data suggest that the ubiquitination system is involved in PCSK9-induced LDLR degradation.

Characterization of the expression and activity of carboxylesterases 1 and 2 from the beagle dog, cynomolgus monkey, and human.

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.