Biomedical subjects

Sheryl L Stamer

Publications and source records attributed to Sheryl L Stamer.

4 recordsLinked to original sources

PubMed2006

Redox instability induced by 4-hydroxy-2-nonenal in porcine and bovine myoglobins at pH 5.6 and 4 degrees C.

Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.

Aldehydes↗

PubMed2006

Selectively increased oxidative modifications mapped to detergent-insoluble forms of Abeta and beta-III tubulin in Alzheimer's disease.

Deleterious post-translational modifications (PTMs) to the neuronal cytoskeleton are a proposed mechanistic link between accumulation of amyloid (A) beta peptides and subsequent abnormalities of tau and neurodegeneration in Alzheimer's disease (AD). Here we tested the hypothesis that PTMs on neuronal tubulins selectively accumulate in a pathological protein fraction in AD. We used new software, P-MOD, to identify comprehensively and map PTMs using mass spectral data from soluble (normal) and detergent-insoluble (pathological) protein fractions from AD, as well as total extracts from controls, for selected proteins: Abeta, tau, apolipoprotein (apo) E, glial fibrillary acidic protein (GFAP), alpha-III tubulin, and beta-III tubulin. Our results confirmed direct observations of others by identifying methionine (M) sulfoxides at Abeta position 35 and numerous sites of tau phosphorylation in detergent-insoluble protein from AD, while no PTMs were enriched on primarily astrocyte-derived apoE or GFAP in this fraction. P-MOD mapped several abundant M sulfoxides to neuron-enriched beta-III tubulin but not its heterodimeric partner, neuron-enriched alpha-III tubulin, a result confirmed by selective suppression of CNBr-mediated cleavage of beta-III tubulin. These findings are the first comprehensive assessment of PTMs in AD and point to oxidative modification of beta-III tubulin as a potential contributor to the neuronal cytoskeletal disruption that is characteristic of AD.

Alzheimer Disease↗

PubMed2005

Sample preparation and digestion for proteomic analyses using spin filters.

We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a > or = 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.

Bicarbonates↗

PubMed2004

Global shifts in protein sumoylation in response to electrophile and oxidative stress.

Human small ubiquitin-like modifier (sumo) proteins include sumo-1 and the less studied, nearly identical sumo-2 and sumo-3 proteins. Whereas the structurally related ubiquitin molecule targets proteins for degradation, sumo provides a distinct, yet poorly understood regulatory signal. Protein sumoylation is sensitive to diverse cellular stresses, yet the targets of sumoylation in stress are unknown. We studied protein sumoylation in HEK293 cells exposed to hydrogen peroxide, alkylating agents, and the lipid oxidation-derived electrophile 4-hydroxynonenal, which is an ubiquitous product of lipid oxidation associated with oxidative stress. Confocal immunofluorescence microscopy indicated that in unstressed cells sumo-1 targeted nuclear proteins, whereas sumo-2/3 targeted proteins in both nuclei and cytoplasm. Western blot analyses revealed changes in sumo-1 and sumo-2/3 targeting patterns with stress. We used immunoaffinity chromatography to harvest sumo-associated proteins from HA-sumo-1- and HA-sumo-3-expressing HEK293 cells both before and after treatment with 4-hydroxynonenal. Multidimensional liquid chromatography-tandem mass spectrometry analyses identified 54 HA-sumo-1-associated proteins and 38 HA-sumo-3-associated proteins. Major protein targets included RNA binding and processing proteins, transcription factors, metabolic enzymes, and cytoskeletal regulators. Treatment with 4-hydroxynonenal caused a near-complete redistribution of sumo-1 and sumo-3 to different protein targets, which included chaperones, antioxidant, and DNA damage signaling proteins. A 10-15% overlap of sumo-1 and sumo-3 targets before and after stress suggests that sumo proteins target distinct protein groups. The results suggest that reactive electrophiles not only directly modify proteins but also lead to indirect changes in endogenous protein modifications that regulate protein functions.

Aldehydes↗