13 research outputs found
Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis
The
most abundant proteins on high-density lipoproteins
(HDLs),
apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of
HDL function with 15 and 9 proteoforms (chemical-structure variants),
respectively. The relative abundance of these proteoforms in human
serum is associated with HDL cholesterol efflux capacity, and cholesterol
content. However, the association between proteoform concentrations
and HDL size is unknown. We employed a novel native-gel electrophoresis
technique, clear native gel-eluted liquid fraction entrapment electrophoresis
(CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate
this association. Pooled serum was fractionated using acrylamide gels
of lengths 8 and 25 cm. Western blotting determined molecular diameter
and intact-mass spectrometry determined proteoform profiles of each
fraction. The 8- and 25 cm experiments generated 19 and 36 differently
sized HDL fractions, respectively. The proteoform distribution varied
across size. Fatty-acylated APOA1 proteoforms were associated with
larger HDL sizes (Pearson’s R = 0.94, p = 4 × 10–7) and were approximately
four times more abundant in particles larger than 9.6 nm than in total
serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide
proAPOA1. APOA2 proteoform abundance was similar across HDL sizes.
Our results establish CN-GELFrEE as an effective lipid–particle
separation technique and suggest that acylated proteoforms of APOA1
are associated with larger HDL particles
An Essential Role for Alzheimer’s-Linked Amyloid Beta Oligomers in Neurodevelopment: Transient Expression of Multiple Proteoforms during Retina Histogenesis
Human amyloid beta peptide (Aβ) is a brain catabolite that at nanomolar concentrations can form neurotoxic oligomers (AβOs), which are known to accumulate in Alzheimer’s disease. Because a predisposition to form neurotoxins seems surprising, we have investigated whether circumstances might exist where AβO accumulation may in fact be beneficial. Our investigation focused on the embryonic chick retina, which expresses the same Aβ as humans. Using conformation-selective antibodies, immunoblots, mass spectrometry, and fluorescence microscopy, we discovered that AβOs are indeed present in the developing retina, where multiple proteoforms are expressed in a highly regulated cell-specific manner. The expression of the AβO proteoforms was selectively associated with transiently expressed phosphorylated Tau (pTau) proteoforms that, like AβOs, are linked to Alzheimer’s disease (AD). To test whether the AβOs were functional in development, embryos were cultured ex ovo and then injected intravitreally with either a beta-site APP-cleaving enzyme 1 (BACE-1) inhibitor or an AβO-selective antibody to prematurely lower the levels of AβOs. The consequence was disrupted histogenesis resulting in dysplasia resembling that seen in various retina pathologies. We suggest the hypothesis that embryonic AβOs are a new type of short-lived peptidergic hormone with a role in neural development. Such a role could help explain why a peptide that manifests deleterious gain-of-function activity when it oligomerizes in the aging brain has been evolutionarily conserved
A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA‑I Proteoforms in Individuals with High and Low HDL Efflux Capacity
Top-down proteomics (TDP) allows
precise determination/characterization of the different proteoforms
derived from the expression of a single gene. In this study, we targeted
apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein
cholesterol efflux (HDL-E), which is inversely associated with coronary
heart disease risk. Absolute ApoA-I concentration and allelic variation
only partially explain interindividual HDL-E variation. Therefore,
we hypothesize that differences in HDL-E are associated with the abundances
of different ApoA-I proteoforms. Here, we present a targeted TDP methodology
to characterize ApoA-I proteoforms in serum samples and compare their
abundances between individuals. We characterized 18 ApoA-I proteoforms
using selected-ion monitoring coupled to electron-transfer dissociation
mass spectrometry. We then compared the abundances of these proteoforms
between two groups of four participants, representing the individuals
with highest and lowest HDL-E values within the Chicago Healthy Aging
Study (<i>n</i> = 420). Six proteoforms showed significantly
(<i>p</i> < 0.0005) higher intensity in high HDL-E individuals:
canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated
ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated
forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated
(fd = 2.31) and one bearing two modifications: palmitoylation and
truncation (fd = 2.13). These results demonstrate translational potential
for targeted TDP in revealing, with high sensitivity, associations
between interindividual proteoform variation and physiological differences
underlying disease risk
Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability
Free fatty acids (FFAs) have been shown to produce alteration of heart rate variability (HRV) in healthy and diabetic individuals. Changes in HRV have been described in septic patients and in those with hyperglycemia and elevated plasma FFA levels. We studied if sepsis-induced heart damage and HRV alteration are associated with plasma FFA levels in patients. Thirty-one patients with sepsis were included. The patients were divided into two groups: survivors(n = 12) and nonsurvivors (n = 19). The following associations were investigated: (a) troponin I elevation and HRV reduction and (b) clinical evolution and HRV index, plasma troponin, and plasma FFA levels. Initial measurements of C-reactive protein and gravity Acute Physiology and Chronic Health Evaluation scores were similar in both groups. Overall, an increase in plasma troponin level was related to increased mortality risk. From the first day of study, the nonsurvivor group presented a reduced left ventricular stroke work systolic index and a reduced low frequency (LF) that is one of HRV indexes. The correlation coefficient for LF values and troponin was r(2) = 0.75 (P < 0.05). All patients presented elevated plasma FFA levels on the first day of the study (5.11 +/- 0.53 mg/mL), and this elevation was even greater in the nonsurvivor group compared with the survivors (6.88 +/- 0.13 vs. 3.85 +/- 0.48 mg/mL, respectively; P < 0.05). Cardiac damage was confirmed by measurement of plasma troponin I and histological analysis. Heart dysfunction was determined by left ventricular stroke work systolic index and HRV index in nonsurvivor patients. A relationship was found between plasma FFA levels, LFnu index, troponin levels, and histological changes. Plasma FFA levels emerged as possible cause of heart damage in sepsis
An informatic framework for decoding protein complexes by top-down mass spectrometry
Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy for hierarchical top-down analysis, identification, and scoring of multi-proteoform complexes by native mass spectrometry.The authors thank members of the Kelleher research group and Prof. V. Wysocki for helpful discussions and advice. O.S.S. is supported by a U. S. National Science Foundation Graduate Research Fellowship (2014171659). P.C.H. is a recipient of a Northwestern University's Chemistry of Life Processes Institute Postdoctoral Fellowship Award. L.H.F.D.V. is supported under CNPq research grant 202011/2012-7 from the Brazilian government. H.S.S is supported under the Science Without Borders scholarship 88888.075416/2013-00 from the Coordination for the Improvement of Higher Education Personnel, under the Brazilian government. This work was supported by grants from the W.M. Keck Foundation (DT061512) and the U.S. National Institutes of Health (GM067193) to N.L.K
Synthesis, cytotoxic activity, and mode of action of new Santacruzamate A analogs.
Breast and ovarian cancer are the most common cancers in women. Available cancer treatments, in general, have limited efficacy and frequent, undesirable side effects. Recently, scientists have focused on searching for new epigenetic modulators such as inhibitors of DNA methyltransferases and histone deacetylases (HDACs), with novel properties and selectivity. We report the synthesis of seven new analogs of Santacruzamate A. Molecular modeling showed that compounds 3?9 presented the best binding energies (kcal/mol) against HDAC4 compared to that of crystallographic ligand. The compounds were evaluated against MCF-7 and MDA-MB-231 (breast cancer), TOV-21G (ovarian adenocarcinoma), and WI-26VA4 (non-tumor lung fibroblasts) cells. Compound 5, the most potent and selective of the series, exhibited remarkably enhanced anticancer potency, with IC50 values for the tumor cells of 24.3?44.93??M, compared with that of etoposide (12?18.57??M) and doxorubicin (2.1?4.37??M). Further investigation showed that compound 5 could promote DNA damage, increase the activity of caspases-3 and -9, and upregulate mRNA levels of p21, TP53, and BAK, suggesting apoptotic cell death of the tumor cells via the intrinsic pathway. This study demonstrated that synthetic analogs of santacruzamate A with zinc-linked groups are effective for improving both HDAC inhibition and antitumor activity