Thesis advisor: Eranthie Weerapana / Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

During postmortem metabolism, hydrogen ions accumulate in the muscle and gradually lower the pH from 7.2 to an ultimate pH near 5.6. The ultimate pH of meat is widely valued as an indicator of fresh meat quality as it directly affects the quality characteristics of color, texture, and water holding capacity. Therefore, our research was conducted to identify the processes responsible for determining ultimate pH. Pigs harboring the AMPK�•3R200Q mutation produce meat with extremely low ultimate pH (pH ~ 5.3) that is detrimental to quality. This phenomenon is often attributed to a greater glycogen content in muscle from the mutant pigs compared to wild-type pigs. However, our research indicated that greater glycolytic flux in muscle from these pigs causes a lower ultimate pH rather than greater tissue glycogen deposition. On the other hand, however, AMPK�•3R200Q pigs contain more mitochondria and retain greater oxidative capacity. Hence, we hypothesized that mitochondria may contribute to the lower ultimate pH in muscle of these pigs. To test our hypothesis, isolated mitochondria were incorporated into an in vitro system the mimics postmortem glycolysis. Mitochondria enhanced glycolytic flux and pH decline in the in vitro system similar to that of AMPK�•3R200Q pigs. After a series of experiments, we found that the causative agent for enhanced glycolytic flux is a soluble mitochondrial protein. In other experiments, mitochondrial F1-ATPase was found to be responsible for the majority of this effect, principally through promoting greater ATP hydrolysis at lower pH values, thereby allowing for greater flux through glycolysis. These data suggest that variations in ultimate pH may be more thoroughly explained and predicted by the abundance of mitochondria. Broiler pectoralis major muscle, which is a highly glycolytic muscle, possesses high ultimate pH (pH ~ 5.9) compared to pork and beef. We postulated that rapid carcass chilling reduces the flux through glycolysis, thereby causing premature termination of postmortem metabolism. Yet, chilling was only partially responsible for the high ultimate pH of pectoralis major muscle. However, we showed that pectoralis major of broiler chicken exhibits lower phosphofructokinase-1 activity compared to porcine longissimus lumborum muscle, which limits the flux through glycolysis. / Ph. D. / Consumer demand for high quality meat has increased dramatically over the past two decades. In order to meet this demand, it is crucial to understand factors that control the development of fresh meat quality characteristics. Consumers purchase meat based on color, but repeat purchases are also influenced by meat freshness, texture, and juiciness. These quality attributes develop after the animal has been harvested during the conversion of muscle to meat through a series of biochemical reactions. This conversion results in muscle acidification (pH decline) caused by the degradation of stored muscle energy that acidifies muscle. Normally, the muscle pH drops from a neutral value in living muscle to an acid value (~5.6) in fresh meat. In some cases, however, excessive acidification occurs and this can dramatically impact fresh meat quality characteristics. Our research program focuses on the mechanisms responsible for this extended acidification. To that end, we use mutant pigs known as RN– that produce meat with extremely low ultimate pH known as “acid meat”. While most believe that the extended pH decline in muscle of these pigs is a function of elevated energy in the muscle prior to harvesting, we showed that theses pigs have different muscle prior to harvest and this difference cause increased acidification during the transformation of muscle to meat. To investigate this issue further, we also examined the contribution of mitochondria (the powerhouse of the cell) to this process mainly because muscle from the RN– pigs containing around 50% more mitochondria compared to normal pigs. Curiously, we have shown mitochondria participate in this process. Because mitochondria require oxygen to function and harvesting animals disrupts oxygen delivery to the muscle, mitochondria were considered irrelevant to the development of meat quality characteristics. Our studies have definitively proved that mitochondria can contribute to meat quality and may be key in improving fresh meat quality.

Type 2 Diabetes Mellitus (T2DM), a metabolic disorder denoted by elevated blood glucose levels and insufficient insulin action, is growing in prevalence worldwide . Barriers to improving disease outcome resolve primarily around identifying and intervening during the preliminary stages of insulin resistance, a state clinically referred to as pre-diabetes. Emerging evidence suggests that mitochondrial dysfunction may underlie , and potentially precede, progressive insulin resistance, suggesting that biomarkers indicative of mitochondrial dysfunction could predict disease risk and status. In this study, we examined epigenetic modifications, in the form of DNA methylation, in the promoter region of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α), a known regulator of mitochondrial biogenesis. Following the initiation of a high fat diet, we observed significant genotypic (DNA methylation) and phenotypic (mitochondrial copy number) alterations in C57/BL6 rodent models. These changes preceded overt disease onset, as classified by clinically utilized indices, which included the homeostatic model assessment for insulin resistance (HOMA-IR), the homeostatic model assessment for β-cell dysfunction (HOMA- β), and the quantitative insulin-sensitivity check index (QUICKI). Our data indicate that methylation analysis may serve as an effective clinical parameter to use in conjunction with physiological criterion for the diagnosis of pre-diabetes and the assessment of T2DM disease risk, and adds to the growing body of work seeking to elucidate the role. / Doctor of Philosophy / High blood glucose, referred to as type 2 diabetes (T2DM), increases the risk for heart and kidney disease, blindness, stroke, and death. Efforts to prevent T2DM have centered primarily around behavioral interventions, which include increased physical activity and decreased caloric intake. Importantly, the interventions are most effective when implemented early on in disease progression. In this study, we sought to examine the effects of a high fat diet on the epigenetic profile of PGC-1α, a gene responsible for maintaining mitochondrial biogenesis. The mitochondria, the powerhouse of the cell, is responsible for maintaining the energy systems in the body. Therefore, we examined how increasing in caloric intake resulted in changes in the epigenetic profile of the PGC-1α promoter, and how these changes impacted mitochondrial number. Further, we sought to examine how hypermethylation of PGC-1α led to changes in gene and protein expression in the mitochondria. Results from our study indicate that DNA methylation changes preceded disease onset, as characterized by the homeostatic model assessment for insulin resistance (HOMA-IR), the homeostatic model assessment for β-cell dysfunction (HOMA- β), and the quantitative insulin-sensitivity check index (QUICKI). Our data indicate that methylation analysis may serve as diagnostic and risk assessment tool for pre-diabetes and T2DM in conjunction with physiological measures.

Sporadic Inclusion Body Myositis (sIBM) is an inflammatory muscle disease that strikes individuals at random and accounts for approximately 1/3 of all idiopathic inflammatory myopathies. It is characterized by progressive weakness of distal and proximal muscles and is the most common muscle disorder in individuals over 50 years of age. Currently, there is no known cause, cure, or enduring treatment for sIBM, although a number of theories as to its cause have been proposed. One theory proposes that activation of the inflammatory/ immune response is the primary trigger resulting in muscle degeneration and protein abnormalities, while an alternative theory suggests that sIBM is a degenerative muscle disease with abnormal pathogenic protein accumulation, in particular Abeta, being a primary cause that triggers an inflammatory/ immune response. Mitochondrial abnormalities have been observed in skeletal muscle from patients diagnosed with the disease, however the role of the mitochondria in disease pathology is still unclear. The aim of this dissertation was to evaluate: 1) the role of the mitochondria in the development of sIBM and 2) the role of amyloid beta on mitochondrial function in skeletal muscle. A better understanding of the role of the mitochondria in the development of sIBM may help to identify novel prevention and/ or treatment strategies. / Ph. D.

Inflammation is a central feature of various metabolic diseases including obesity and type-II diabetes. For this study, we hypothesized postprandial metabolism following an acute, high fat (HF) meal to be impaired in mice pre-injected with an inflammatory agonist. To this end, C57BL/6J mice were injected with saline or lipopolysaccharide (LPS, 1μg/kgbw) following an overnight fast and gavaged 2hr post-injection with water or a HF meal in liquid form (5kcal; 21.4%SF, 40.8%UF, 27.1%CHO, 10.7%PRO). Blood and muscle samples taken 3hr post-gavage underwent ex vivo analysis. Overall, results demonstrated a metabolic response to a HF meal that was blocked in the presence of LPS. Metabolic flexibility, though unchanged following the HF meal alone, was reduced following the HF meal in the presence of LPS. Additionally, state-4 uncoupled mitochondrial respiration, which was increased following the HF meal, was also reduced following the HF meal in the presence of LPS. Similar near-significant trends were also observed with total palmitate oxidation. Although no independent response to a HF meal or LPS exposure was observed, a unique interaction between treatments significantly diminished ADP dependent, state-3 and maximal respiration. These effects do not appear to be dependent on the production of reactive oxygen species (ROS) since neither the HF meal nor LPS exposure resulted in increased production of ROS. In conclusion, these results demonstrate that acute activation of inflammatory pathways results in alterations in metabolic response to a HF meal in skeletal muscle from mice, although the mechanism underlying these effects is not yet understood. / Master of Science

Yes / Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic. / A.R.V., M.W., and T.E. were supported by the US Army Research Office (W911NF-19-1-0235). L.S., M.W., and T.E. were supported by the United Mitochondrial Disease Foundation (PI-19-0985). L.S. was also supported by the University of Exeter Jubilee Scholarship. M.C., N.J.S., and T.E. were supported by the UK Space Agency (ST/R005737/1). N.J.S. and T.E. were supported by BBSRC (BB/N015894/1). S.A.V. was supported by NASA (NNX15AL16G). N.J.S. was supported by grants from NASA [NSSC22K0250; NSSC22K0278] and acknowledges the support of the Osteopathic Heritage Foundation through funding for the Osteopathic Heritage Foundation Ralph S. Licklider, D.O., Research Endowment in the Heritage College of Osteopathic Medicine.

Mitochondria play a central role in lipid metabolism and pathology in obesity and type 2 diabetes mellitus. Mitochondria have been shown to associate with lipid droplets (LDs) in multiple tissues but the functional role of these peridroplet mitochondria (PDM) is unknown. This work reveals that PDM have unique protein composition and cristae structure, and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities. (2) PDM have reduced beta oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and beta oxidation. (3) PDM support LD expansion as Perilipin 5-induced recruitment of mitochondria to LDs increases ATP-dependent triacylglyceride synthesis. (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis. We suggest that increased mitochondrial recruitment to LDs may be part of a generalized adaptive response in physiological conditions that require LD expansion, such as post-prandial lipid synthesis and storage. Furthermore, PDM-mediated LD expansion may play a role in muscle and liver injury from lipotoxicity in conditions of nutrient excess, such as obesity and hyperlipidemia. A better understanding of PDM and LD biology may therefore lead to new therapies for lipotoxic tissue injury and insulin resistance. / 2020-10-24T00:00:00Z

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis. / Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus.