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Age-associated alterations in endoplasmic reticulum function
We have strong theoretical and preliminary evidence suggesting that decay of endoplasmic reticulum-dependent protein processing and attendant stress sensing mechanisms may be a major reason for the decline. The ER represents a major nexus where protein processing, cellular redox state, and signaling pathways intertwine and ensure proper protein processing and trafficking. Aging leads to disruption of this concerted process, resulting in chronic disturbances in regulatory and signaling proteins of the ER proteome. These alterations lead to down-regulation of ER stress response pathways and increase the overall vulnerability to internal and external stress stimuli. We are currently utilizing a rodent model of aging to test our hypothesis using the following approaches:
  • Characterize the age-related changes in oxidation states of major redox metabolites, protein sulfhydryls, and resident and client protein load.

  • Identification of specific ER stress signaling pathways that are altered with age.

  • Determining the consequences of age-related changes in ER function contributes to vulnerability to environmental stresses (e.g. toxicologic exposures, high fat diet, and infection)
Small molecule metabolite profiling for accurate in vivo assessment of oxidative and inflammatory stress (with Mark Shigenaga and Bruce Ames)
Oxidants cause damage to biological macromolecules and disrupt cellular homeostasis. Aminothiols are sensitive targets of oxidation and represent a first line of defense against various types of oxidants. These antioxidant defenses decline in disease-states, in part, through the oxidant-induced conversion to their respective disulfide forms. Changes in levels and oxidation states of glutathione (GSH) have primarily been used as a measure of intracellular thiol redox state. Focus on GSH has been, in part, due to limits in the sensitivity of assays aimed at detection of other less abundant aminothiols.

We developed a sensitive liquid chromatography/tandem mass-spectrometry (LC/MS/MS) method for the detection and quantification of oxidation states of a comprehensive panel of aminothiols. A strong cation exchange solid phase extraction procedure is used to concentrate the acid-soluble aminothiols in biological samples. Isopropylchloroformate (IPCF) is added to rapidly (2-3 min) esterify the reactive sulfhydryl, carboxyl and amino groups. IPCF modifies the free thiol groups at low pH ranges (1-6) and provides a more precise determination of thiol redox states. The IPCF method in conjunction with LC/MS/MS instrumentation improves detection sensitivity and selectivity of aminothiols. Detailed redox profiles of aminothiols (e.g., homocysteine, cysteine, cysteinylglycine, and glutathione) are readily obtained using this method. In addition, the current method is flexible and readily modified to detect other non-thiol amino acids, which provides the capacity to target of multiple metabolic pathways of interests. For example, many of the metabolites involved in the Urea cycle and the polyamine synthesis pathways can be easily detected in a single chromatographic run.

This new technique has significant potential as a diagnostic tool in various diseases like autism, sickle cell disease, thalassemia, polycystic ovarian syndrome and childhood obesity.


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