Alumni Dissertations

 

Alumni Dissertations

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  • BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF DI-ASPARTYL INTRAMEMBRANE PROTEASES

    Author:
    Celia Mirtha Torres Arancivia
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Iban Ubarretxena
    Abstract:

    A new era in the study of proteases was initiated with the discovery of intramembrane-cleaving proteases (I-CLiPs). In an unusual mechanism, I-CLiPs are able to catalyze peptide bond cleavage of substrates within the membrane bilayer, an environment poorly permeable to water. Here we will focus on gamma-secretase and presenilin(PS)-like proteases, membrane proteases that use two aspartic acids as catalytic residues and constitute the most relevant family of I-CLiPs. We first describe the purification of gamma-secretase from a mammalian expression system. We used this preparation for negative-stain single-particle electron microscopy and determined the structure of a native-like catalytically active gamma-secretase complex at a resolution of 25 Å. Antibody labeling of the extracellular domain of Nicastrin was employed to ascertain the topology of the reconstruction. In addition, active site labeling with a gold-coupled inhibitor demonstrated that gamma-secretase contains a single active site facing a large conical internal cavity. Secondly, we used the yeast P. pastoris as an expression system to isolate milligram quantities of active and fully mature gamma-secretase. The higher yield of gamma-secretase obtained from this system allowed the addition of a size exclusion chromatography step to the purification procedure to reduce the structural heterogeneity of the preparation and thereby improve the structural analyses. Finally, we provide novel proof for the existence of a presenilin-like protease in the euryarchaeota M. marisnigri (MCMJR1). We tested in vitro protease activity on chimeric protein substrates containing transmembrane domain (TMD) regions and found that MCMJR1 was indeed able to generate a proteolytic product. Mutagenesis confirmed that two aspartic acid residues were essential for the activity of the protease. Also, the activity of MCMJR1 was blocked when an inhibitor of gamma-secretase was incubated with the enzyme. Mass spectrometry analysis revealed that MCMJR1 cleaves the chimeric substrates close to the middle of the TMD with promiscuous peptide bond selectivity. In conclusion, our study has enhanced the understanding of PS/gamma-secretase and provided new routes for functional and structural studies on di-aspartyl intramembrane proteases, both of which are pivotal in unraveling the mechanistic details of intramembrane proteolysis in biology and disease.

  • A coarse-grained view of protein-protein recognition

    Author:
    Rodney Versace Babilonia
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Marco Ceruso
    Abstract:

    One of the most important characteristics of proteins is their ability to specifically interact with other proteins and with other types of molecules to build supramolecular assemblies in order to perform different kinds of functions. Protein-protein interaction has been a subject of study in several sciences including biochemistry, structural biology, and computational biophysics/biology. One limitation that delays our understanding of molecular recognition is the lack of high-resolution three-dimensional structures of the protein-protein complexes. Because of this, the methods for computational prediction have gained popularity and importance but in many cases, the predicted complex is not accurate. Predicting the native three-dimensional conformation of protein-protein complexes still remains a big challenge. Most biological processes in the cell involve a huge number of atoms and happen at time-scales that are frequently beyond the current limits of classical atomistic simulations. The ELNEDIN approach is a new and powerful coarse-grained representation with the ability to overcome size and time limits without deforming the overall shape of a protein. Previous studies have shown that the quality of the ELNEDIN scaffold influences the ability of the modeled proteins to experience structural transitions and to associate and form a stable complex. The main objective of this thesis is to test if the ELNEDIN approach is able to discriminate native interfaces from non-native. The Barnase/Barstar complex, the RNase/Barnase complex and the Ubiquitin/Ubiquitin ligase complex were chosen to test this hypothesis. Each individual protein model was simulated using the ELNEDIN approach and the potential of mean force for the dissociation of the complex was calculated. Our results show: 1) It is possible to obtain accurate energy-profiles using the ELNEDIN approach. 2) The shape of the free energy landscape around a protein receptor has a funnel-like shape where the bottom of the funnel is the global minimum and it starts to increase smoothly. 3) The solvent plays an important role in the shape of the free energy profile. 4) The ELNEDIN approach is able to recognize the native conformation for hydrophilic interfaces. To further support the last result, an additional complex was chosen: the Nuclease A/Nuclease inhibitor A complex.

  • Identification and characterization of protein phopshatases regulating the Sma/Mab pathway in C. elegans

    Author:
    Sheng Xiong
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Cathy Savage-Dunn
    Abstract:

    Identification and characterization of protein phosphatases regulating the Sma/Mab signaling pathway in C. elegans by Sheng Xiong Advisor: Professor Cathy Savage-Dunn TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved TGFβ pathway, the Sma/Mab pathway. We used a C. elegans RNA interference library of phosphatases to identify genes that cause a body size phenotype. Library-wide screening was carried out in an RNAi-hypersensitive mutant background, rrf-3. To further narrow the candidate pool, we analyzed the body size phenotypes of these candidates using different genetic backgrounds. These analyses allow us to narrow the candidate pool down to 80-candidates. A Sma/Mab pathway reporter, RAD-SMAD reporter (a kind gift from Dr. Jun Liu, Cornell University), was used to assess whether the candidates regulate body size phenotype in a Sma/Mab pathway-dependent manner or not. The reporter assay revealed 22 likely candidates regulate Sma/Mab signaling directly or indirectly. Among them, 11 candidates were verified as protein phosphatases by sequencing. These include homologues of mammalian PPM1A/B/G and PP1. Our study revealed that ppm-1 (metal-ion dependent protein phosphatase-1), a human PPM1A/B/G homolog, might act as a potential SMA-3-specific linker phosphatase to regulate the Sma/Mab pathway. This is the first time to show that a homolog of human PPM1A/B/G might act as a potential linker phosphatase of R-Smads to promote TGFβ signaling. Our studies also showed that a homolog of the inhibitory regulatory subunit of human PP1 might synergize with the catalytic subunit in the regulation of TGFβ signaling for the first time. We also discovered the regulatory role of several protein tyrosine phosphatases in this signaling cascade. In summary, this study sheds a light on elucidating the regulatory mechanism of TGFβ signaling pathway, therefore providing insight in various TGFβ signaling-involved human developmental disorders and cancer, and contributing to the development of potential diagnostic markers and therapeutic targets in human diseases.

  • Investigate the Effects of Osteogenesis Imperfecta Mutations on the Conformation of Collagen Triple Helix

    Author:
    Ke Xu
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Yujia Xu
    Abstract:

    The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the _1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly3Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen. In addition to the biomedical studies, the recombinant collagen fragments have also proven to be a good system to produce nano-templates. The rigidity of the helix backbone, the linear conformation, and the largely exposed side chains of residues at the X and Y positions for chemical modification make the triple helix an ideal template for nanowires

  • The role of the Epstein Barr Virus Nuclear Antigen-1 in the production of antibodies to dsDNA

    Author:
    Pragya Yadav
    Year of Dissertation:
    2012
    Program:
    Biochemistry
    Advisor:
    Linda Spatz
    Abstract:

    Previous studies have shown an association between the Epstein Barr Virus (EBV) and the development of the autoimmune disease, Systemic Lupus Erythematosus (SLE). However, it has not yet been proven that EBV plays a causative role in the etiology of SLE. In the present study, I demonstrate that mice injected with the major EBV nuclear protein, EBNA-1, can develop antibodies to double stranded DNA (dsDNA), which are the hallmark of SLE. To understand the basis for the anti-dsDNA response, I generated monoclonal antibodies (MAbs) to EBNA-1 from EBNA-1 injected mice. I made the novel observation that some of these MAbs cross-react with dsDNA. One of these MAbs, designated 3D4, was shown to bind to the glomeruli of mouse kidneys. This is a feature of the pathogenic anti-dsDNA antibodies in lupus, which can deposit in the kidney and cause renal damage (nephritis). In an effort to map the epitope in EBNA-1 that elicits cross-reactivity to dsDNA, I generated several truncated fragments of the EBNA-1 protein and examined the binding of the cross-reactive MAbs to these fragments. All of the cross-reactive MAbs that I examined recognized an epitope that resides within amino acids 459 and 607 in the carboxyl region of EBNA-1. This 148 amino acid region is confined to the viral binding site (VBS) of EBNA-1 and contains a well-defined secondary structure, although, it is not yet known whether the epitope is linear or conformational. We are currently trying to map this epitope further to define a smaller peptide that these MAbs recognize. Identification of a small epitope that serves as a peptide mimic for dsDNA may help in the design of diagnostic strategies for screening and therapeutic strategies for treating patients with SLE.

  • ACTIVITY AND REGULATION OF CYCLIN-DEPENDENT KINASE 5 (CDK5) DURING CELL DEATH

    Author:
    Yixia Ye
    Year of Dissertation:
    2009
    Program:
    Biochemistry
    Advisor:
    Zahra Zakeri
    Abstract:

    ACTIVITY AND REGULATION OF CDK5 DURING CELL DEATH Cell death is an important element of development, tissue maintenance and disease. This biological process is tightly controlled by different cell death signaling pathways and a number of genes have been implicated in the regulation of cell death. Cyclin-dependent kinase 5 (Cdk5), identified due to its sequence homology to Cdc2, is a gene that is activated during cell death. The aim of this study is to investigate the roles of Cdk5 by examining the relationship of Cdk5 to different pathways involved in cell death, then evaluate the regulation of Cdk5 activation during cell death and whether Cdk5 activity is required for the induction of cell death. By using different models in which massive cell death was induced by different toxins, including cyclophosphamide (CP)-treated mouse embryos, cyclohexamide (CHX) or camptothecine (CPT)-treated mouse embryonic fibroblast cell lines and measuring the modulation of different cell death related genes, such as caspase-3, calpain and lysosomal proteases, cathepsins, we have shown that the activation of Cdk5 is dispensible of the activity of caspase-3, calpain, and the lysosomal proteases, cathepsins. Furthermore, the finding that Cdk5 is activated without the generation of p25, an activator of Cdk5 during cell death, suggests that other activators for Cdk5 activation exist during cell death. Additionally, inhibition of the production of Cdk5 activators, p25 and p29, by calpain inhibitor or down-regulation of Cdk5 activity by RNAi reveals Cdk5-independent cell death; and the mode of cell death is not altered in these situations. We therefore conclude that Cdk5 activation during cell death is a result of cell death rather than an initiator of cell death.

  • Temporal proteasome disruption has a long term and irreversible negative impact on drosophila

    Author:
    Chun-Hung Yeh
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Thomas Schmidt-Glenewinkel
    Abstract:

    The presence of unfolded, misfolded, mutant and/or oxidized proteins is a severe and never ending threat to cell survival. To prevent these abnormal proteins to form aggregates and disrupt cellular homeostasis, these proteins can be delivered to the ubiquitin proteasome system (UPS) for degradation. To investigate the proteotoxic effects induced by a dysfunctional UPS, we developed a Drosophila model with "tunable" proteasome impairment by modulating the expression of one of its catalytic subunits, the dâ5 subunit, through double-stranded RNA interference (RNAi). Expression of the dâ5 RNAi is controlled by an RU486 inducible Act5C promoter. Reduction of dâ5 subunit expression is caused by RU486 administration. The loss of dâ5 function causes an early onset of reduced proteasome activity. Disruption of proteasome activity has negative impacts including ubiquitinated-protein accumulation, a shorter lifespan, low resistance to oxidative stress, and locomotor dysfunction. Many UPS-impaired models are established to address effects caused by proteasome dysfunction. However, no model investigates whether temporal proteasome disruption causes long term or irreversible effects. To address this issue, temporal proteasome disruption was induced by varying the period of RU486 administration via the conditional Gene-Switch binary system. Our results demonstrate that temporal proteasome disruption causes long term/irreversible and negative effects on lifespan, protein degradation, elimination of aggregates, and locomotor activity. Taken together, these results indicate that UPS plays critical roles in maintenance of cellular function. Proteasome disruption, even just temporally, has a long term and negative impact in drosophila.

  • DBL-1/BMP target genes and sma-9 function in C. elegans

    Author:
    Jianghua Yin
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Cathy Savage-Dunn
    Abstract:

    Transforming growth factor β (TGF-β) signaling regulates a large number of biological processes including cell proliferation, lineage determination, differentiation, motility, adhesion, and death. In Caenorhabditis elegans, the DBL-1 pathway, a BMP-related signaling pathway, regulates body size and male tail development. A Drosophila Schnurri homolog, sma-9, acts as a transcription cofactor in the DBL-1 pathway (Liang et al., 2003). By comparing gene expression profiles of sma-9 and dbl-1 mutants to wild type animals, we found that DBL-1/BMP signaling pathway regulates a large number of target genes including collagen genes, transcription factors, genes involved in innate immunity, dauer and aging genes (Liang et al., 2007). Here, we have verified a series of potential target genes and their involvement in body size regulation. T27F2.4, a bZip transcription factor, is expressed in intestine and neurons, is negatively regulated by DBL-1/BMP signaling and inhibits growth. col-41, a type IV cuticle collagen gene, is expressed in hypodermis, is positively regulated by DBL-1/BMP signaling and promotes normal growth. col-41 promoter analysis indicates that the first 500bp promoter region is essential for its basal level expression, but does not contain the cis-regulatory elements responsible for sma-9 regulation. The regulation of col-41 by sma-9 in vivo could be a combination of direct and indirect effects. In addition to T27F2.4 and col-41, we have also identified other target genes responsive to DBL-1/BMP signaling, which greatly expands the potential toolkit of reporters for further analysis of DBL-1/BMP signaling. The sma-9 locus in vivo undergoes alternative splicing, including an unusual trans-splicing event that could generate two non-overlapping shorter transcripts. We have demonstrated that the shorter transcripts are expressed in vivo. Furthermore, we find that one of the short transcripts plays a tissue-specific role in sma-9 function, contributing to the patterning of male-specific sensory rays, but not to the regulation of body size. Based on previous results, we suggest that this transcript encodes a C-terminal SMA-9 isoform that may provide transcriptional activation activity, while full length isoforms may mediate transcriptional repression.

  • MECHANISMS OF DEADENYLATION REGULATION UNDER DIFFERENT CELLULAR CONDITIONS

    Author:
    Xiaokan Zhang
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Frida Kleiman
    Abstract:

    Control of gene expression by regulating mRNA stability after DNA damage has the potential to contribute to the cells rapid response to stress. The main focus of this dissertation is to elucidate the role(s) of nuclear PARN deadenylase in controlling mRNA stability, hence gene expression, of factors in the p53 signaling pathway during the DNA damage response (DDR). Understanding the mechanisms of these regulatory pathways will provide new insights on how the control of gene expression upon DNA damage decides cellular fate, offering new opportunities for therapeutics. In Chapter II, I presented evidence that PARN along with the cleavage factor CstF-associated tumor suppressor BARD1 participates in the regulation of endogenous transcripts in different cellular conditions. In Chapter III, I identified the mRNA targets of PARN in non-stress conditions, and contributed to describing a feedback loop between p53 and PARN, in which PARN deadenylase keeps p53 levels low by destabilizing p53 mRNA through its 3Oe untranslated region (3fUTR) in non-stress conditions, and the UV-induced increase in p53 activates PARN, regulating gene expression during DDR in a transactivation-independent manner. In Chapter IV, I presented evidence that PARN deadenylase has a specific effect on the steady-state levels of not only AU-rich element-containing but also microRNA (miRNA)-regulated nuclear mRNAs. I showed that the functional interaction of PARN with miRNA-induced silencing complex contributes to p53 mRNA stability regulation. These studies provide the first description of PARN deadenylase function in miRNA-dependent control of mRNA decay and of miRNA-function in the nucleus. Finally, in Chapter V, I determine that nucleolin is one of the RNA binding proteins that recruits PARN to the p53 mRNA and this can be regulated by phosphorylation, representing a novel regulatory mechanism for p53 gene expression. The data presented in this dissertation has contributed to describe and comprehend some novel mechanisms behind the regulation of gene expression during DDR.