ABSTRACT
Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a ß-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.-Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.
Subject(s)
Amyloid/chemistry , Apolipoprotein A-I/chemistry , Lipoproteins, HDL/chemistry , Methionine/chemistry , Amyloid/metabolism , Apolipoprotein A-I/metabolism , Atherosclerosis/metabolism , Atherosclerosis/pathology , Humans , Lipoproteins, HDL/metabolism , Methionine/metabolism , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Peroxidase/chemistry , Peroxidase/metabolismABSTRACT
High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.
Subject(s)
Amyloid/metabolism , Apolipoprotein A-I/metabolism , Atherosclerosis/metabolism , Peroxidase/metabolism , Plaque, Atherosclerotic/metabolism , Amyloid/genetics , Apolipoprotein A-I/genetics , Atherosclerosis/genetics , Atherosclerosis/pathology , Humans , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Methionine/genetics , Methionine/metabolism , Oxidants/pharmacology , Oxidation-Reduction/drug effects , Peroxidase/genetics , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/pathologyABSTRACT
Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.
Subject(s)
Carcinoma, Hepatocellular , Cyclic AMP-Dependent Protein Kinases , Humans , Cyclic AMP-Dependent Protein Kinases/metabolism , Glycogen Synthase Kinase 3/metabolism , Carcinoma, Hepatocellular/genetics , Signal Transduction , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Cell Line, TumorABSTRACT
T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.
ABSTRACT
PURPOSE: To construct a wellness committee (WC) implementation index and determine whether this index was associated with evidence-based intervention implementation in a workplace health promotion program. DESIGN: Secondary data analysis of the HealthLinks randomized controlled trial. SETTING: Small businesses assigned to the HealthLinks plus WC study arm. SAMPLE: Small businesses (20-200 employees, n = 23) from 6 low-wage industries in King County, Washington. MEASURES: Wellness committee implementation index (0%-100%) and evidence-based intervention implementation (0%-100%). ANALYSIS: We used descriptive and bivariate statistics to describe worksites' organizational characteristics. For the primary analyses, we used generalized estimating equations with robust standard errors to assess the association between WC implementation index and evidence-based intervention implementation over time. RESULTS: Average WC implementation index scores were 60% at 15 months and 38% at 24 months. Evidence-based intervention scores among worksites with WCs were 27% points higher at 15 months (64% vs 37%, P < .001) and 36% points higher at 24 months (55% vs 18%, P < .001). Higher WC implementation index scores were positively associated with evidence-based intervention implementation scores over time (P < .001). CONCLUSION: Wellness committees may play an essential role in supporting evidence-based intervention implementation among small businesses. Furthermore, the degree to which these WCs are engaged and have leadership support, a set plan or goals, and multilevel participation may influence evidence-based intervention implementation and maintenance over time.
Subject(s)
Health Promotion , Occupational Health , Workplace , Adolescent , Adult , Aged , Female , Humans , Leadership , Male , Middle Aged , Randomized Controlled Trials as Topic , Small Business , Washington , Young AdultABSTRACT
Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti-amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A-I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid-like aggregates formed by methionine-oxidized (ox) apoA-I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of ß-structure in ox-apoA-I amyloid-like aggregates formed in the presence of curcumin, compared to aggregates formed without curcumin. However, the kinetics of ox-apoA-I amyloid-like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox-apoA-I amyloid-like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA-I and alters the structure of ox-apoA-I amyloid-like aggregates yet does not diminish the propensity of ox-apoA-I to form aggregates.