Biomedical subjects

Ruth Hogue Angeletti

Publications and source records attributed to Ruth Hogue Angeletti.

8 recordsLinked to original sources

PubMed2006

Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18.

The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.

Carbon Isotopes↗

PubMed2006

Automatic deconvolution of isotope-resolved mass spectra using variable selection and quantized peptide mass distribution.

We present an algorithm for the deconvolution of isotope-resolved mass spectra of complex peptide mixtures where peaks and isotope series often overlap. The algorithm formulates the problem of mass spectrum deconvolution as a classical statistical problem of variable selection, which aims to interpret the spectrum with the least number of peptides. The LASSO method is used to perform automatic variable selection. The algorithm also makes use of the quantized distribution of peptide masses in the NCBInr database after in silico trypsin digestion as filters to aid the deconvolution process. Errors in the expected isotope pattern are accounted for to avoid spurious isotope series. The effectiveness of the algorithm is demonstrated with annotated ESI spectrum of known peptides for which the peaks and isotope series are highly overlapping. The algorithm successfully finds all correct masses in the experimental spectrum, except for one spectrum where an additional refinement procedure is required to obtain the correct results. Our results compare favorably to those from a widely used commercial program.

Animals↗

PubMed2006

Endogenous transforming growth factor-beta receptor-mediated Smad signaling complexes analyzed by mass spectrometry.

ASmad proteins are the central feature of the transforming growth factor-beta (TGF-beta) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-beta were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-beta was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-beta. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-beta signal transduction processes.

Amino Acid Sequence↗

PubMed2005

Structural and functional characterization of glycosylation in an immunoglobulin G1 to Cryptococcus neoformans glucuronoxylomannan.

Analysis of the N-linked oligosaccharides of the murine IgG1 monoclonal antibody (mAb) to Cryptococcus neoformans by LC/MS revealed five different core fucosylated, biantennary complex-type oligosaccharides at Asn-293, with the major species being a mono-galactosylated oligosaccharide with the glycosyl composition of Hex4HexNAc4Fuc (39% of the total glycan pool). The primary sequence predicted from nucleic acid sequencing differed from that measured by mass spectrometry at position 33 (ASN to ASP), a finding that may represent post-translational modification caused by spontaneous ASP deamination. Analysis of mAb 18B7 from three hybridoma clones revealed the same heterogenous N-glycan pattern, indicating that diversity in oligosaccharide structures originated from individual cells. The binding of native and de-glycosylated mAb 18B7 to cryptococcal Ag was comparable but the de-glycosylated 18B7 had shorter serum half-life and did not activate complement (C). De-glycosylated mAb 18B7 was opsonic for C. neoformans with murine macrophages through a mechanism that involved C-independent ingestion through the C receptor. Passive administration of de-glycosylated mAb 18B7 mediated comparable protective efficacy to the native mAb in mice with lethal infection. The results imply that the contribution of N-glycan structure to immunoglobulin function varies depending on the Ag-Ab system.

Amino Acid Sequence↗

PubMed2005

Interaction with PDZK1 is required for expression of organic anion transporting protein 1A1 on the hepatocyte surface.

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

Amino Acid Sequence↗

PubMed2003

Direct analysis of tubulin expression in cancer cell lines by electrospray ionization mass spectrometry.

Differential expression of tubulin isotypes, mutations, and/or post-translational modifications in sensitive and Taxol-resistant cell lines suggests the existence of tubulin-based mechanisms of resistance. Since tubulin isotypes are defined by their C-terminal sequence, we previously described a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of tubulin diversity in human cell lines by analysis of their CNBr-released C-terminal peptides [Rao, S., Aberg, F., Nieves, E., Horwitz, S. B., and Orr, G. A. (2001) Biochemistry 40, 2096-103]. We now describe the liquid chromatography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein. Four major isotypes, betaI-, betaIVb-, Kalpha1-, and alpha6-tubulin, were detected in two human carcinoma cell lines, A549 and HeLa. betaIII-Tubulin represented a minor species, as did alpha4-tubulin which was detected for the first time in both cell lines. The three alpha-tubulins were almost totally tyrosinated, and post-translational modifications were limited to low levels of monoglutamylation of Kalpha1-, betaI-, and betaIII-tubulin. betaII- and betaIVa-tubulins were not detected in either parental or drug-resistant cell lines, in contrast to previous RNA-based studies. Since mutations can occur in a single tubulin allele, the question as to whether the wild-type and mutant transcripts are both translated, and to what levels, is important. Heterozygous expression of Kalpha1- or betaI-tubulin mutants that introduced mass changes as small as 26 Da was readily detected in native tubulins isolated from Taxol- and epothilone-resistant cell lines.

Amino Acid Sequence↗

PubMed2003

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry.

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.

Amino Acid Sequence↗

PubMed2002

Acute elevation of NEFA causes hyperinsulinemia without effect on insulin secretion rate in healthy human subjects.

Increased circulating levels of nonesterified free fatty acids (NEFA) have been observed in such hyperinsulinemic states as obesity, impaired glucose tolerance, diabetes, and dyslipidemia where they have been causally linked to the development of insulin resistance and hyperinsulinemia. The concentration of NEFA in plasma is believed to have direct modifying effects on insulin secretion and clearance. It remains controversial whether acute increases in NEFA potentiate insulin secretion in human subjects. We studied the effect of an acute elevation of NEFA during lipid-heparin infusion compared to a glycerol-only control on glucose-stimulated insulin secretion and clearance during a 120-min hyperglycemic (10 mM) clamp in 7 healthy normoglucose-tolerant volunteers. The metabolic clearance rate of C-peptide (MCR(CP)) was measured in each subject during the study by simultaneous infusion of C-peptide. Insulin secretion rate (ISR) was calculated from deconvolution of C-peptide data after correction for the rate of C-peptide infusion. Clearance rate of insulin (MCR(INS)) was calculated based upon endogenous ISR. Plasma glucose (mg/dL): basal (90-115 min) 90.2 +/- 2.8 vs. 90.2 +/- 2.3; clamp (150-240 min) 180.5 +/- 2.8 vs. 180.9 +/- 1.3. Plasma insulin (pmol/L): prebasal (fasting) 29.6 +/- 10.0 vs. 29.8 +/- 10.6; basal (90-115 min) 30.1 +/- 9.2 vs. 34.5 +/- 12.1; second phase clamp (210-240 min) 127.6 +/- 18.2 vs. 182.5 +/- 17.3*. Plasma NEFA (mM): prebasal 0.47 +/- 0.08 vs. 0.52 +/- 0.09; basal 0.35 +/- 0.05 vs. 0.98 +/- 0.02*; clamp (122-240 min) 0.06 +/- 0.02 vs. 0.77 +/- 0.06*. ISR (pmol/min): prebasal 72.7 +/- 7.5 vs. 72.0 +/- 7.9; second phase clamp (210-240 min) 268.5 +/- 27.2 vs. 200.2 +/- 23.7. MCR(INS) (mL/min): prebasal 3393 +/- 488 vs. 3370 +/- 511; clamp 2284 +/- 505 vs. 1214 +/- 153* (*p < 0.05 glycerol vs. intralipid/heparin). This study demonstrates that acute NEFA elevation causes hyperinsulinemia due to a significant decrease in systemic insulin clearance without increasing rates of insulin secretion.

Adult↗