A Systematic Investigation of Structure/Function Requirements for The Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu159–Leu170) in nascent HDL, the so-called “solar flare” (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861–868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro165, Tyr166, Ser167, and Asp168) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr166 was further supported using reconstituted HDL generated from apoA-I mutants (Tyr166 → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr166-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr166-apoA-I, after subcutaneous injection into hLCATTg/Tg, apoA-I−/− mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency

Site-Specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr166), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr166 nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr166-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr166-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼8% of total apoA-I within the artery wall but was nearly undetectable (\u3e100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr166-apoA-I existed as a lipid-poor lipoprotein with \u3c3% recovered within the HDL-like fraction (d = 1.063–1.21). NO2-Tyr166-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr166-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr166 is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall

A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein

The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in highdensity lipoprotein (HDL) maturation.Wepreviously identified a highly solvent-exposed apoA-I loop domain (Leu¹⁵⁹–Leu¹⁷⁰) in nascent HDL, the so-called “solar flare” (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861–868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro¹⁶⁵, Tyr¹⁶⁶, Ser¹⁶⁷, and Asp¹⁶⁸) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr¹⁶⁶ was further supported using reconstituted HDL generated from apoA-I mutants (Tyr¹⁶⁶→Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO₂-Tyr¹⁶⁶-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr¹⁶⁶-apoA-I, after subcutaneous injection into hLCATᵀᵍ/ᵀᵍ, apoA-I_/_ mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent- exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency

Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host

Transmission of Atherosclerosis Susceptibility with Gut Microbial Transplantation

Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility

Overcoming EGFR T790M and C797S resistance with mutant-selective allosteric inhibitors

EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Multifunctional, inexpensive, and reusable nanoparticle-printed biochip for cell manipulation and diagnosis

Isolation and characterization of rare cells and molecules from a heterogeneous population is of critical importance in diagnosis of common lethal diseases such as malaria, tuberculosis, HIV, and cancer. For the developing world, point-of-care (POC) diagnostics design must account for limited funds, modest public health infrastructure, and low power availability. To address these challenges, here we integrate microfluidics, electronics, and inkjet printing to build an ultra-low-cost, rapid, and miniaturized lab-on-a-chip (LOC) platform. This platform can perform label-free and rapid single-cell capture, efficient cellular manipulation, rare-cell isolation, selective analytical separation of biological species, sorting, concentration, positioning, enumeration, and characterization. The miniaturized format allows for small sample and reagent volumes. By keeping the electronics separate from microfluidic chips, the former can be reused and device lifetime is extended. Perhaps most notably, the device manufacturing is significantly less expensive, time-consuming, and complex than traditional LOC platforms, requiring only an inkjet printer rather than skilled personnel and clean-room facilities. Production only takes 20 min (vs. up to weeks) and $0.01-an unprecedented cost in clinical diagnostics. The platform works based on intrinsic physical characteristics of biomolecules (e.g., size and polarizability). We demonstrate biomedical applications and verify cell viability in our platform, whose multiplexing and integration of numerous steps and external analyses enhance its application in the clinic, including by nonspecialists. Through its massive cost reduction and usability we anticipate that our platform will enable greater access to diagnostic facilities in developed countries as well as POC diagnostics in resource-poor and developing countries