Khalil Abnous. Ph.D. Chemistry 2007

Regulation of metabolic enzymes during hibernation in ground squirrels



Hibernation is a winter survival strategy for many small mammals. Animals sink into deep torpor, body temperature falls to near 0°C and physiological functions are strongly suppressed. Enzymes are the catalysts of cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of key enzymes of carbohydrate metabolism (hexokinase, HK), energy metabolism (creatine kinase, CK; AMP deaminase, AMPD) and signal transduction (Akt; MAPKAP-K2), highlighting skeletal muscle ground squirrels (Spermophilusrichardsonii). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, mRNA transcript and protein levels, and binding to myofibrils are involved in regulating these enzymes during hibernation. Moreover, reversible protein phosphorylation proved to be a key regulatory mechanism, reducing the activity of all these enzymes during hibernation. Analysis of total protein content by Western blotting found decreased HKII, CK and P-Akt protein during hibernation but no change in Akt and MAPKAP-K2 content. Analysis of temperature effects on enzymes, via Arrhenius plots, showed that CK, AMPD and MAPKAP-K2 had significantly higher activation energies in hibernating animals Urea denaturation and pulse proteolysis showed that HKII from hibernators had greater resistance to chemical denaturation than the euthermic enzyme but studies on CK and MAPKAP-K2 found no stability differences. Affinity of CK and AMPD for their substrates decreased during hibernation. HK, Akt and MAPKAP-K2 showed reduced ATP affinity in hibernation but HK affinity for glucose remained stable, and Akt and MAPKAP-K2 showed higher affinity for their substrate peptides. Protein kinases (PKA, PKC, PKG) increased AMPD activity from both euthermic and hibernating animals but decreased CK activity; AMPK elevated HK activity in euthermic muscle. Protein phosphatases generally reversed these actions. Changes in enzyme phosphorylation state during hibernation were confirmed by elution profiles of the enzymes off DEAE Sephadex, patterns that were interconverted after incubations that stimulated protein kinases and phosphatases. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing enzymatic function and stability during hibernation.


Christopher Dieni.  Ph.D. Chemistry 2008

Regulation of enzyme function in freeze tolerance



The wood frog (Rana sylvatica) is one of the few vertebrate species that can survive whole-body freezing during the cold winter months. The frog endures the freezing of 65-70% of total body water as extracellular ice and, while frozen, shows no respiration, heart beat, or brain activity. Consequently, the frogs experience anoxia and ischemia throughout the freeze followed by oxidative stress when oxygen is reperfused. Enzymes, the biochemical catalysts of cells, must be appropriately controlled to ensure survival. This thesis explores the properties and regulation of key enzymes of adenylate metabolism (AMP-deaminase, AMPD; creatine kinase, CK) and glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH; hexokinase, HK). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, and binding to myofibrils are involved in regulating these enzymes in the transition to the frozen state. Moreover, reversible protein phosphorylation appears to be a key regulatory mechanism, altering enzyme activity and substrate affinity to suit physiological needs during freezing. Analysis of kinetic parameters showed an increase in enzyme activity for CK and decreased activity for HK. Affinity of CK for one of its substrates, creatine, increased, whereas HK, G6PDH, and myofibril-bound AMPD showed reduced substrate affinity in the transition to the frozen state. These changes in kinetic parameters were the result reversible protein phosphorylation; bound AMPD and CK both increased in phosphorylation state in frozen frogs, whereas G6PDH and HK both decreased in phosphorylation state. Changes in enzyme activity as a result of reversible phosphorylation were analyzed by in vitro stimulation of endogenous protein kinase and protein phosphatase activities. Native phosphorylation states of these enzymes, and changes between control and frozen frogs were further confirmed by elution profiles off DEAE-Sephadex ion-exchange columns that were interconverted between the two physiological states, as well as SDS-PAGE studies that compared phosphoprotein levels to total protein levels. Though phosphorylation states of these enzymes changed, protein levels remained constant in the transition to the frozen state. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly reversible protein phosphorylation, control enzyme function and the reorganization of metabolic pathways in freeze-tolerance.

Amal I. Malik, Ph.D. Biology, 2009

Cellular adaptation to dehydration stress: Molecular adaptations for dehydration tolerance in the African clawed frog, Xenopus laevis



The thesis addressed multiple questions about the signal transduction mechanisms that trigger gene expression responses to dehydration signals, and about the role of antioxidant defenses in combating dehydration stress in the African clawed frog, Xenopus laevis. In the first part of the thesis the responses to dehydration stress by the three main MAPK cascades were traced by measuring both total protein levels, and the relative amounts of active phosphorylated proteins for multiple intermediates in the p38 MAPK, stress-activated protein kinase (SAPK), and extracellular signal-regulated kinase (ERK) cascades. The data documented a major activation of the ERK pathway in most organs of X. laevis during dehydration. Selected upstream activator and downstream targets of the ERK pathway also showed pronounced tissue specific regulation in response to dehydration. The SAPK was activated in skeletal muscle, lung, and skin whereas the p38 MAPK was activated in the lung and kidney of X. laevis.  The second part of the thesis focused on antioxidant defenses that are known to be contributors to cell preservation under various types of stress. Two main transcription factors that regulate antioxidant genes were activated in response to dehydration in X. laevis organs:  NF-E2 related factor (Nrf2) and forkhead box, class O (FoxO). Immunoblottingshowed a significant increase in their nuclear translocation, and enzyme-linked immunosorbant assays showed increased DNA binding activity by FoxO1 under dehydration stress. Expression of downstream target genes controlled by these transcription factors was enhanced during dehydration. Six family members of the glutathione S-transferase (GST) and three family members of the aldo-keto reductase (AKR) showed tissue specific expression, correlated with Nrf2 activation. Manganese-dependent superoxide dismutase (MnSOD) and catalase expression were also elevated under FoxO1 control. Improved antioxidant defenses may be critical to dealing with variations in tissue oxygenation and reactive oxygen species generation that are one consequence of large changes in body hydration that affect oxygen delivery to tissues. This thesis showed for the first time that the MAPKs family members are selectively activated in response to two levels of dehydration stress in X. laevis. Also, antioxidant defenses play a critical protective role during dehydration stress in these frogs.

Anastasia Krivoruchko, Ph.D. Biology, 2010

Turtle anoxia – biochemistry and gene regulation in an anaerobic extremist



While the physiological responses to oxygen deprivation have been studied extensively in the anoxia-tolerant turtle, Trachemysscripta elegans, adaptations of transcriptional regulatory processes are mostly unknown. This thesis addresses this by examining the anoxia responsiveness of several important proteins and pathways in T. s. elegans tissues. The unfolded protein response (UPR) was activated in turtle heart, kidney and liver, as evidenced by increased phosphorylation of PERK and increased expression and activation of ATF4. Enhanced expression of the molecular chaperones GRP78 and GRP94, as well as other UPR-responsive proteins was also observed. These results suggest that the UPR is an important component of stress tolerance in the turtle. The transcription factor NF-kB was also anoxia-responsive in turtle liver and activated via increased expression of its component proteins, increased nuclear presence and increased DNA-binding activity. Transcript levels of NF-kB target genes involved in antioxidant defense and anti-apoptotic signaling were also upregulated under anoxia. The FoxO transcription factors, implicated in hypometabolism and stress resistance, were also anoxia-responsive. Studies of FoxO expression, phosphorylationstatus, nuclear presence and DNA-binding activity showed that FoxO1 and FoxO3 were both activated in liver, whereas FoxO3 was activated in heart and kidney. FoxO target genes involved in cell cycle arrest and stress resistance were also upregulated in liver under anoxia. Expression and activation of the transcriptional inhibitors, histone deacetylases (HDACs), was strongly elevated in white skeletal muscle during anoxia, with a lesser response by liver, results that indicated an important role for HDACs in anoxia-mediated transcriptional suppression. Finally, the metabolic transcription factor involved in control ofglycolytic enzymes, ChREBP, was activated in the liver in response to 5 h of anoxia, and its target gene, LPK, wastranscriptionaly induced, suggesting a role for this transcription factor in adjusting carbohydrate metabolism for anaerobiosis. Overall, the data in this thesis enhance understanding of the gene and protein adaptations that support cellular endurance of anoxia and document several new mechanisms that are involved in stress resistance, hypometabolism and fuel metabolism as being key to natural anoxia tolerance.

Benjamin Lant, Ph.D. Biology, 2011

Expression pattern of the novel freeze-responsive genes li16, fr10 and fr47 in the wood frog, Rana sylvatica



The capacity to adapt to and survive oxygen deprivation has long been an important topic of study, in both ecological and medical fields. The freshwater crayfish, Orconectes virilis, is capable of tolerating anoxia, but the metabolic mechanisms underlying this are largely unprofiled. This thesis examines the activity and regulation of a number of stress response pathways in response to anoxia in O. virilis. The model organism Caenorhabditis elegans that shows stress-induced entry into hypometabolism (the dauer stage) was used as a template for selecting stress response pathways that could be important in crayfish anaerobiosis. The Akt signaling response showed a distinct increase in activity in crayfish tail muscle and hepatopancreas under anoxia, as assessed through phosphorylation states of the kinase and its downstream targets. This implicated a pro-survival response that functions by preventing cell cycle attenuation. Despite elevated Akt activity, residualFoxO activity remained, possibly mediating a pro-survival mechanism through transactivation of antioxidant genes (includingMnSOD) in preparation for reoxygenation. Smad and STAT transcription factors, following the pattern of pro-development Akt signaling, also showed a fairly active profile (via phosphorylation status) but upregulation was not unilateral. Hepatopancreas showed a more active profile of Smads, but this did not correlate with increased DNA binding, again hinting at a preparative mechanism for the recovery period. Apoptosis (cell death) signaling was assessed through pro-apoptosis (p53) and anti-apoptosis (Bcl) targets, whereas autophagy (a cell minimization response to stress) was assessed via expression response of multiple autophagy proteins (Atg). An anoxia-triggered, tissue-specific result arose, potentially based on the importance of individual organ integrity throughout hypometabolism. Tail muscle, which showed increased expression profiles of all three target groups (p53, Bcls, Atgs), contrasted with hepatopancreas, which appeared to be not susceptible to either apoptotic orautophagic signaling during anoxia. Finally, the cell cycle, often a target for attenuation in stress states, was analyzed. Neither tissue showed strong signs of cell cycle attenuation under anoxia, although certain inhibitor profiles were enhanced under anoxia. The data provide a comprehensive overview of the responses and integration of multiple stress-responsive signaling pathways in O. virilis that provide a novel contribution to our understanding of pro-survival mechanisms supporting invertebrate anoxia tolerance.