Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms.

Development of the nervous system is a highly organized process that utilizes genetic mechanisms conserved across the animal kingdom. Components of the nervous system such as inhibitory GABAergic neural networks are common among most multicellular animals. The nematode Caenorhabditis elegans, utilizes similar genetic pathways to that of mice and humans to develop its GABAergic neural networks. These GABAergic neural networks are composed of two types of GABAergic motor neurons: the VD and DD sub-classes. The GABAergic differentiation of both these sub-classes requires the conserved transcription factor, Pitx/UNC-30. The VD sub-class is differentiated from the DD motor neurons by the expression of another transcription factor, COUP TFII/UNC-55. The transcriptional mechanisms regulating the expression of Pitx/UNC-30 and Coup TFII are unknown. We sought to determine how Pitx/UNC-30 and COUP TF-II/UNC-55 were transcriptionally regulated in an attempt to understand how mechanisms of GABAergic fate specification and class specification may be connected. We hypothesized there would be different mechanisms regulating the GABAergic differentiation and sub-class specification of the two sub-classes of GABAergic motor neurons. To test this, we dissected the transcriptional mechanisms responsible for the expression of Pitx/UNC-30 and COUP TFII/UNC-55. We found that different isoforms of the Hox cofactor Meis/UNC-62 stabilize and activate the expression of UNC-55. Furthermore, we conclude that Pitx/UNC-30 expression is regulated differently between the two motor neuron sub-classes by Meis/UNC-62, Hox-B7/MAB-5 and NeuroD/CND-1, each of which are vital to the development of different components of the nervous system in vertebrates. Our findings suggest that the GABAergic identity and the sub-class specification of neurons are under the control of multiple conserved transcription factors responsible for neuron fate determination and post mitotic identities.

Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...

La sénescence réplicative (SR) dans les fibroblastes humains primaires est causée par l’érosion des télomères et est contrôlée par p53. La régulation dynamique de l’activation de p53 est essentielle pour l’induction de la sénescence ; cependant, les mécanismes moléculaires sous-jacents ne sont pas clairement établis. Nous montrons, dans les cellules surexprimant les isoformes Δ133/Δ160p53, que ces isoformes s’oligomérisent avec p53, conduisant ainsi à la stabilisation d’une forme inactive de p53. A l’inverse, l’inactivation des isoformes endogènes Δ133/Δ160p53 induit l’accumulation de la protéine p53 et l’activation de son activité transcriptionnelle. La surexpression de Δ133/Δ160p53 inhibe les fonctions de p53, en particulier son activité transcriptionnelle et son rôle dans l’arrêt du cycle après un dommage à l’ADN. Nous avons remarqué que les protéines Δ133/Δ160p53 et p53 sauvage possédaient des conformations différentes. Les protéines Δ133/Δ160p53 sont reconnues pas l’anticorps Pab40 : elles adopteraient une conformation similaire à un mutant de conformation de p53. Enfin, nous observons qu’une faible expression de l’ARNm Δ133/Δ160TP53 coïnciderait avec la durée de l’activation transcriptionnelle de p53 lors de la SR, indiquée par l’accumulation de l’ARNm d’un effecteur majeur de p53, p21. L’augmentation de l’expression de Δ133/Δ160TP53 à un temps tardif au cours de la SR est corrélée à l’accumulation du marqueur de sénescence p16INK4a et à celle de la cytokine pro-inflammatoire IL-6. En conséquence, les isoformes Δ133/Δ160p53 contrôleraient l’activité de p53 dans l’arrêt du cycle et sur le phénotype sécrétoire des cellules sénescentes. / Telomere attrition in primary human fibroblasts induces replicative senescence by activation of the tumour suppressor p53. Fine-tuned activation of p53 is essential for senescence induction; however, the mechanisms underlying the regulation of p53 activity during senescence have not been clearly established yet. We report here that in cells that express the Δ133/Δ160p53 isoforms, these p53 isoforms oligomerize with p53, leading to the stabilization of the transcriptionally inactive form of p53. Conversely, endogenous Δ133/Δ160p53 silencing increases the level of p53 and p53-dependent transcriptional activity to promote cell cycle arrest. Overexpressed Δ133/Δ160p53 repress p53 functions, including gene transcription activation and growth inhibition, upon DNA damage. We also found that Δ133/Δ160p53 and wild-type p53 have different structural conformations. Δ133/Δ160p53 adopt a more unfolded conformation recognized by the Pab240 antibody, indicating that these p53 isoforms have a p53 mutant-like conformation. Finally, we observed that low level of Δ133/Δ160TP53 mRNA coincided with the duration of p53 transcriptional activation in replicatively senescent fibroblasts, as indicated by the upregulation of CDKN1A (p21) mRNA expression, a downstream effector of p53. Δ133/Δ160p53TP53 was upregulated at a later stage when the senescence marker p16INK4a and the pro-inflammatory interleukin-6 (IL-6) were also induced. Therefore, p53 activity on growth suppression and senescence-associated secretory phenotype may be differentially regulated by its Δ133/Δ160p53 isoforms.

The human proteom diversity is caused by the ability of a single gene locus to encode more protein isoforms. The TCF/LEF genes produce a broad spectrum of protein variants, which consequently leads to a great functional diversity of the TCF/LEF proteins. The TCF/LEF transcriptional factors regulate the canonical Wnt signaling target genes. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1

INTRODUCTION: Retinal Pigment Epithelia (RPE) degeneration is a key event in the development of age-related macular degeneration (AMD). RPE dysfunction in AMD is thought to occur through the accumulation of reactive oxygen species (ROS) and oxidative damage. The transcriptional co-activators, PGC-1α and PGC-1β, are important regulators of mitochondrial biogenesis and anti-oxidant capacity. Our group has previously shown that the PGC-1α protein promotes RPE oxidative metabolism and that overexpression of the PGC-1α gene protects cells from AMD-associated pro-oxidants. On the other hand, PGC-1β gene expression has been found to be upregulated in patients with neovascular AMD, and in-vitro overexpression of PGC-1β damages cells and induces pro-oxidant conditions. OBJECTIVE: Given the divergence of PGC-1α and PGC-1β functions in RPE and their clinical relevance in AMD pathogenesis, this project will seek to investigate the impact of the upregulation of PGC-1α and PGC-1β in RPE metabolism. PGC-1α will be upregulated through treatment with compound ZLN005. A new methodology for PGC-1β expression will be developed to closely modulate in-vitro PGC-1β induction. METHODS: In-vitro experiments were performed on the ARPE-19 cell line. Cells were treated with 10µM of ZLN005 for 24 hours. Oxidative stress was induced by exposure to H2O2 and NaIO3 under serum-free conditions. Lactate dehydrogenase (LDH) levels were used to quantify cell death. Quantitative PCR (qPCR) and Western Blot were performed to measure changes in gene and protein expression respectively. Superoxide production by the mitochondria was measured to evaluate ROS levels within the cell. Intravitreal injections of 20µM ZLN005 were performed on eight-week old male C57BL/6J mice. After 24 and 72 hours of treatment, the mice were euthanized and the enucleated eyes were dissected to obtain the RPE and neural retina layers. Total RNA was extracted from these layers and qPCR was performed to measure gene expression. A tetracycline-inducible PGC-1β plasmid was designed and transfected into ARPE-19 cells. The cells were exposed to 0.01-100µg/ml doxycycline for 48-hours and qPCR was used to measure gene expression. Transfected cells were treated with ZLN005 and cell death upon exposure to oxidative stress was quantified. RESULTS: Gene expression analysis on ARPE-19 cells treated with ZLN005 showed robust upregulation of PGC-1α, PGC-1β and their associated transcription factors and enzymes. Induction of PGC-1α at the protein level was also confirmed. ZLN005 efficiently protected ARPE-19 cells from H2O2 and NaIO3 cytotoxicity and its protection was negated in PGC-1α-silenced cells. Treatment with ZLN005 also decreased mitochondrial superoxide production. ZLN005 intravitreal injections were safely administered to the animals and did not cause cataracts or other damage to the ocular tissues. While statistical significance in gene expression changes was limited due to the small sample size, anti-oxidants GPX1 and TXN2, and electron transport chain gene, ATP50, were found to be potentially induced in the neuro-retina, while FOXO3 was found to be downregulated. Evaluation of our novel tetracycline-inducible PGC-1β adenoviral vector showed that upregulation of PGC-1β was efficiently controlled by the addition of doxycycline to transfected cells. Upon exposure to H2O2, transfected cells treated with doxycycline experienced greater cell death than transfected cells not exposed to doxycycline. ZLN005 treatment was able to decrease cell death in both conditions. CONCLUSION: The present study shows that ZLN005 efficiently protects RPE cells from oxidative damage through selective induction of PGC-1α. While still preliminary, the in-vivo study indicates that ZLN005 is safe to be injected into the eye and may be able to increase the expression of mito-protective and anti-oxidant genes in the neuronal retina. In addition, our design of the tetracycline inducible PGC-1β plasmid allows for tight control over PGC-1β expression through doxycycline addition. Upregulation of PGC-1β at levels similar to those observed in clinical conditions caused increased pro-oxidant induced cell death and treatment with ZLN005 was able to protect against cell death. / 2021-06-30T00:00:00Z

Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.

<p>Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. </p><p>PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP.</p><p>Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.</p>

Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP. Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

An array of analytical methods including density gradient ultracentrifugation, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were utilized to analyze serum high density lipoprotein (HDL) subfractions from two cohorts of normolipidemic individuals, which included subjects with diagnosed coronary artery disease (CAD), and angiographically proven non-CAD controls. These methods collectively provided characteristic information about the two populations of individuals including composition, electrophoretic mobilities, molecular weights, isoforms, and post-translational modifications of HDL apolipoproteins. This information proved useful in identifying potential biomarkers for CAD risk, and understanding the biological functions of a novel atherogenic HDL phenotype in individuals with CAD. Through the implementation of the aforementioned methodologies, new isoforms of apoC-I were identified. MALDI-MS, detected a shifting of approximately 90 Da in the mass to charge ratios corresponding to apoC-I peaks in the serum subfractions from all CAD cohort patients. This shifting was not observed in the non-CAD cohort, which displayed apoC-I peaks in accordance with the known mass of this protein. In addition to the shifting observed in the CAD cohort, some CAD patients showed further modifications of apoC-I that were indicative of oxidative processes. Interestingly, one patient, who has not been diagnosed with CAD, and has a family history of the disease, contained the apoC-I isoforms. This feature could underlie this subject’s known family history of CAD, and serve as an initial screening that could indicate the future development of CAD in this individual. Through collaborative work with Johns Hopkins University, it was initially observed that apoC-I enriched HDL induced apoptosis of aortic smooth muscle cells. Conversely, apoC-I depleted HDL induced minimal to no apoptosis, which led to the hypothesis that apoC-I is a contributor to atherogenic HDL and is a potential risk factor for CAD. Further collaborative work with Johns Hopkins assessed the apoptosis levels induced by HDL from both cohorts of patients. A distinct difference in apoptosis was identified between the two cohorts. High density lipoprotein subfractions from subjects in the CAD cohort, all of which contained the apoC-I isoforms, induced marked apoptosis compared to the non-CAD controls. These results further supported the hypothesis that apoC-I compromises the functionality of HDL and showed that through the induction of apoptosis, apoC-I can contribute to the destabilization of atherosclerotic plaque and the acceleration of CAD.