Research Catalyst
Julie A. Reisz Haines, PhD
Research Assistant Professor
Redox regulation of enzymatic activity and metabolism
The scope of my projects is best defined as understanding metabolic adaptations and consequences to altered oxygen status. Before joining the D’Alessandro lab, I was involved in numerous projects investigating the effects of sulfur oxidation on protein structure and function. In particular, I studied the chemical reactivity of cysteine sulfenic acids (Cys-SOH) in efforts to develop new reagents for their detection by mass spectrometry (MS) and to understand the regulation and reversibility of cysteine oxidation in signaling. These interests paired nicely when I joined the group with an ongoing project to investigate protein oxidation in RBCs during storage. We showed in Blood in 2016 (Reisz et al. Blood 2016, 128, e32) that oxidative modifications to GAPDH cysteines impede glycolysis in a storage duration-dependent fashion. Using activity assays and switch-tagging approaches, we showed that early on, cysteines are reversibly modified (e.g., disulfide or sulfenic acid) but end-of-storage results in an enrichment of irreversibly oxidized (e.g., sulfinic or sulfonic acid) GAPDH thiols. We are continuing to investigate this phenomenon in other redox-sensitive RBC proteins and how their modification impacts metabolism.
On the other side of the oxygen continuum, I am involved in several exciting projects aimed at elucidating the effects of and response to hypoxia in different diseases and physiological states. In collaboration with the Trauma Research Center on campus and at Denver Health, we analyzed a timecourse set of plasma samples from 95 trauma patients taken immediately after injury through one week and found that plasma succinate was the strongest correlate with mortality (up 25.9-fold in deceased vs survivors) (D’Alessandro et al. J Trauma Acute Care Surg 2017, 83, 491). We next performed a comparison analysis of four mammals (rat, swine, macaque, and human) to understand the diversity of metabolic responses to hemorrhagic shock, focusing on succinate and lactate in particular (Reisz et al. J Trauma Acute Care Surg 2017, ahead of print):
The theme of hypoxia has also been utilized in our studies of health and disease. In RBC biology, I have been involved in the metabolic assessment of RBCs a) stored under various low oxygen conditions; b) from patients and mouse models of sickle cell disease; c) from patients with pre-eclampsia. I enjoy observing patterns of metabolic and proteomic response to changes in oxygen status and the many ways in which the outcomes are interconnected.
Figure 1 – In A, a structural representation of GAPDH active site pocket, with Cys152 and His179 modified as to show two confidently assigned redox modifications (Cys to DHA; His to 2-oxo-His). In B, ratios of NEM-tagged or IAM-tagged Cys152 (green and red continuous lines, respectively) vs total spectral matches for GAPDH in total RBC extracts at storage days 2, 21 and 42. Results represent a readout of unmodified or reversibly-modified Cys152 GAPDH at lysis, respectively. In C, absolute quantitation of GAPDH in stored RBC supernatants through QconCAT analysis. In D, semi-quantitative determination of oxidative modifications to Cys152 of GADPH are shown, calculated by determining the ratios of modified/total occurrences of confidently observed (p <0.05) GAPDH peptide IISNASCTTNCLAPLAK, divided by the total number of all peptides confidently assigned for GAPDH. In E a representative MS2 spectra of the peptide IISNASCTTNCLAPLAK of human GAPDH (residues 146-162), showing an irreversible modifications of the functional Cys152
Figure 2 – Plasma levels of lactate (mM) and succinate (µM) in rats, pigs, non-human primates and critically ill patients at baseline (green) and shock (red). Mean + standard deviations are indicated. (* p <0.05; ** p <0.01; *** p<0.0001 T-test).