Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Deformed Epidermal Autoregulatory Factor-1 (DEAF-1) is a transcription factor that binds TTCG motifs and has roles in fetal development, clinical depression and cancer. In order to further our understanding of the DEAF-1 protein, this study characterizes previously unidentified DEAF-1 interacting proteins and post-translational modifications of DEAF-1. A region encompassing the DNA binding domain of DEAF-1 interacts with the C-terminal Bax interacting domain of the Ku70 subunit of the DNA-PK holoenzyme. Ku70 acts as an anti-apoptotic protein through C-terminal domain and so DEAF-1 was assessed for its ability to influence apoptosis after various stimuli. DEAF-1 acted as a pro-apoptotic protein after intrinsic stimuli. Apoptotic activities occurred through a nuclear, DNA independent mechanism and a mutation that eliminated Ku70 interactions also inhibited DEAF-1 pro-apoptotic activities. Analysis of mammalian purified DEAF-1 indicated a number of phosphorylation sites and also a methylated arginine residue. Various assays were performed on mutated forms of DEAF-1 to determine the significance of the modified sites on DEAF-1 functions and properties. Lysine mutation of the methylated arginine site appeared to augment protein-protein interactions with itself and also Ku70. Alanine mutations at three of the identified phosphorylation sites increased DEAF-1 pro-apoptotic activities. In vitro kinase assays identified CDK5 as potential kinase that can phosphorylate DEAF-1. These studies provide new insight into potential functions, properties, and regulation of DEAF-1.

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The role of protein kinases in modulating cell proliferation was examined. Studies characterized the regulation of cell proliferation by adenosine 3':5'-monophosphate-dependent protein kinase (cA-PK). Calcium/calmodulin-dependent myosin light chain kinase (MLCK) was isolated and examined as a potential substrate regulated by cA-PK in the rapidly proliferating.

Rapid and well-orchestrated action potential propagation through the myocardium is essential to each heartbeat. Gap junctions comprising primarily Cx43 reside within the intercalated discs connecting cardiomyocytes, effecting not only direct intercellular electrical coupling, but the localization of other junctional structures and ion channels. Alterations in Cx43 expression occur in essentially all forms of heart disease and is therefore a topic of intense study. Posttranslational modification of Cx43 is understood to impact trafficking, conduction, and stability. Altered Cx43 phosphorylation is well described during pathological remodeling of gap junctions in response to cellular stress. Research has revealed how phosphorylation of specific residues elicit specific effects on Cx43, but the complexity of this process has left much unknown. In particular, the role phosphorylation of a triplet of double serine residues, Ser365, Ser368, and Ser373, plays in GJ function and Cx43/14-3-3 interaction has been called into question. Using an ex vivo whole heart ischemia model we find a decrease in pS368 in mice lacking the ability to phosphorylate S365 and S373 while under stress. In vitro transfection of human induced pluripotent stem cell-derived cardiomyocytes when stressed with PMA were also carried out. These data allow us to piece together the exquisite interplay of gatekeeper phosphorylation events upstream of channel closure, altered protein-protein interactions, and gap junction internalization and degradation. It is hoped that our increasing understanding of this important area of gap junction biology will facilitate better understanding of arrhythmogenesis, and potential therapeutic strategies to restore or preserve normal electrical coupling in diseased hearts. / Master of Science / The heart, an electrically active organ, relies on the propagation of an electrical signal throughout its entirety in order to produce a healthy heartbeat. In order to do so, the heart uses specialized muscle cells known as cardiomyocytes which can not only contract but pass along chemical signals to the cardiomyocyte next in line to signal it to contract as well. The passage of signals occurs through protein units called gap junctions and are made predominantly of Cx43 proteins in the heart. Gap junctions look and function like tubes that travel from the inside space of one cell to the other and allow a flow of small molecules to occur; these small molecules, namely ions, are part of the signal needed to initiate contraction in the adjacent cell. Cx43, like many proteins in our bodies, is slightly altered after it is produced through a process known as posttranslational modification. This allows the cell to alter the localization and function of the protein and tailor it for the needs of the cell. Rather than changing the backbone composition of the protein, small chemical groups are attached, and this imparts a change to how the protein interacts with other proteins or its environment. In particular, one form of modification is known as phosphorylation where a phosphate group is attached to the protein at specific locations along its chain. Cx43 too can be phosphorylated, and while under pathological stress, such as a lack of oxygen or infection, cardiomyocytes increase the amount of phosphorylated Cx43 at a site known to cause pathological changes to the function of Cx43. These changes include how well the gap junctions can transmit signals or associate with other proteins and, in the heart, can predispose the development of arrhythmias or unhealthy heartbeats. However, not all phosphorylation is bad and phosphorylation at other locations also occurs during normal healthy functions of the cardiomyocyte can affect how other sites along Cx43 are phosphorylated. The process of one phosphorylated site affecting another is known as the gatekeeper effect and add a new layer to our understanding of how cells use phosphorylated Cx43 to fine tune its effects. Using cells that do not produce their own Cx43 and subsequently giving them the instructions to produce specific forms of mutant Cx43 that can and cannot be phosphorylated at specific sites, we can understand with greater detail of how cardiomyocytes respond to stress and how some of those responses can be pathological. This will allow future research into the creation of therapies that prevent negative Cx43 phosphorylation after illness, potentially avoiding the development of dangerous arrhythmias.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to environmental and physiological stresses, cells impose translational control to reprogram adaptive gene expression and conserve energy and nutrients. A central mechanism regulating translation involves phosphorylation of the a-subunit of the eukaryotic initiation factor -2 (p-eIF2a), which reduces delivery of initiator tRNA to ribosomes and represses global protein synthesis. The pathway featuring p-eIF2a is called the integrated stress response because it involves multiple related eIF2a kinases, each responding to different stress arrangements. While p-eIF2a limits global protein synthesis, a subset of mRNAs are preferentially translated in response to p-eIF2a. Preferential translation of stress adaptive mRNAs is regulated by upstream opening reading frames (uORFs) present in the 5’-leader of these transcripts. In most cases uORFs are inhibitory in nature, but in some case uORFs can instead promote the translation of the downstream CDS. This study is focused on preferential translation of the gene Inhibitor of Bruton’s Tyrosine Kinase-alpha (IBTKa) in response to endoplasmic reticulum stress. The human IBTKa gene encodes a 1353 amino acid residue protein, along with a 5’-leader featuring predicted canonical uORFs. Among the four predicted uORFs, the 5'-proximal uORF1 and uORF2 are phylogenetically conserved among mammals and are well translated as judged by reporter assays, whereas uORF3 and uORF4 are not conserved and are poorly translated. In addition to the uORFs in the IBTKa mRNA, a phylogenetically conserved stem-loop (SL) of moderate stability is present 11 nucleotides downstream of uORF2. Using luciferase reporter assay, the uORF2 and SL were shown to function together to repress the translation of human IBTKa. In non-stressed conditions, the SL combined with uORF2 are critical for reducing ribosomes from reinitiating at the IBTKa coding sequence (CDS), thus repressing IBTKa expression. Upon ER stress and induced p-eIF2a, the more modestly translated uORF1 facilitates the bypass of the inhibitory uORF2/SL to enhance the translation of main CDS of IBTKa. This study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as a “bar code” by which scanning ribosomes decide which mRNAs are preferentially translated in the integrated stress response. / 2023-10-03

The huntingtin N17 domain is the master regulator of huntingtin intracellular localization. N17 is post-translationally modified, and phosphorylation of N17 serines 13 and 16 facilitate the stress dependent nuclear translocation of huntingtin by inhibiting CRM1 binding and nuclear export. In Huntington’s disease (HD), mutant huntingtin is hypo-phosphorylated and increasing N17 phosphorylation has been shown to be protective in HD mouse models. N17 phosphorylation is therefore a valid therapeutic sub-target of huntingtin. The ER stresses that have been previously characterized to affect huntingtin phosphorylation are broad, likely activating a plethora of response pathways. Thus, in this study, we sought to define a specific stress that could affect huntingtin phosphorylation and nuclear localization. Here we show that huntingtin localization and phosphorylation can be specifically affected by reactive oxygen species (ROS). We identify a highly conserved methionine at position 8 (M8) as the specific sensor of oxidative species within N17 and show the capacity of oxido-mimetic M8 point mutations to affect N17 structure, localization and phosphorylation. We also define a specific molecular mechanism whereby N17 oxidation promotes membrane dissociation, thus increasing kinase accessibility and subsequent phosphorylation. These results define a precise molecular mechanism for the normal biological regulation of huntingtin phosphorylation by oxidative signalling. This ability of huntingtin to sense ROS levels at the ER provides a link between age-associated stress and altered huntingtin function. It suggests that ROS stress due to aging may be a critical molecular trigger of HD that could explain the age-onset nature of disease. / Thesis / Master of Science (MSc)