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Uncovering New Mechanisms of Cdc34 and Cullin-Ring Activity

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UNLV Theses, Dissertations, Professional Papers, and Capstones 12-15-2019 Uncovering New Mechanisms of Cdc34 and Cullin-Ring Activity Spencer Hill Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Biochemistry Commons Repository Citation Hill, Spencer, "Uncovering New Mechanisms of Cdc34 and Cullin-Ring Activity" (2019). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3808. http://dx.doi.org/10.34917/18608668 This Dissertation is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. UNCOVERING NEW MECHANISMS OF CDC34 AND CULLIN-RING ACTIVITY By Spencer Wayne Hill Bachelor of Science in Biochemistry University of Nevada, Las Vegas 2013 A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy - Chemistry Department of Chemistry and Biochemistry College of Sciences The Graduate College University of Nevada, Las Vegas December 2019 Dissertation Approval The Graduate College The University of Nevada, Las Vegas November 1st, 2019 This dissertation prepared by Spencer Wayne Hill entitled Uncovering New Mechanisms of Cdc34 and Cullin-Ring Activity is approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy - Chemistry Department of Chemistry and Biochemistry Gary Kleiger, Ph.D. Kathryn Hausbeck Korgan, Ph.D. Examination Committee Chair Graduate College Dean Ernesto Abel-Santos, Ph.D. Examination Committee Member Hui Zhang, Ph.D. Examination Committee Member Laurel Raftery, Ph.D. Graduate College Faculty Representative ii ABSTRACT UNCOVERING NEW MECHANISMS OF CDC34 AND CULLIN-RING ACTIVITY By Spencer Wayne Hill Dr. Gary L Kleiger, Examination Committee Chair Department of Chemistry and Biochemistry University of Nevada, Las Vegas Ubiquitylation is a cellular regulatory system found in all eukaryotic cells, which has managed to find a role in most pathways imaginable. The system works fundamentally through the ligation of a small protein known as ubiquitin onto a substrate. Depending on the context of the ubiquitin ligation, the substrate can be directed towards a number of cellular fates, the best- studied being degradation of the substrate. While originally thought of as a signal for cellular disposal units to degrade aberrant proteins, we now know that ubiquitin plays a highly nuanced role in cellular epistasis, controlling everything from the cell cycle to the immune response. Of the many enzymes involved in transferring ubiquitin molecules to and from targeted substrates, the cullin-RING ubiquitin ligases (CRLs) stand out for their particular breadth. Hundreds of CRLs exist in human cells owing to their modular structure, which in turn allows them to regulate an even greater number of substrates. They have also been targets of a number of different drug therapies, due to their involvement in the cell cycle and cancer. However, there are many vital gaps in how they function. iii Particularly, CRLs function with a number ubiquitylating enzymes, referred to here as CRL partners. The first discovered of these, Cdc34, has been prominently studied for decades, but particular aspects of its molecular mechanism for transferring ubiquitin to substrates were not yet known. Further complexity was added when other CRL partners were discovered to also function in addition to Cdc34. Promising models suggested that these CRL partners could complement the activity of Cdc34 to maximize CRL turnover of substrate, but relatively little work had been done to study this system under the consideration of physiological conditions and concentrations. Therefore, the central aims of the studies within the planned dissertation are three-fold. First, by designing and refining current assays to set a guideline by which to measure complex, multi-component reactions. Second, by uncovering the molecular mechanism of Cdc34’s catalytic activity, so that it can be understood in the context of full CRL activity. Lastly, by determining how CRLs and their partners operate in the context of a living cell. For these efforts, we discovered a new molecular mechanism of Cdc34 activity, a new understanding of how CRL partners are balanced and used in the context of physiological CRL ubiquitylation pathways, and the unexpected complementary role of a little-studied CRL partner. iv ACKNOWLEDGEMENTS First I have to thank Dr. Kleiger, who has been my advisor since I first became interested in scientific research as an undergraduate. My initial plans in college were to go down the route of patent law upon reception of my Bachelor’s, but I quickly discovered that working in his lab was far too rewarding to stop. There have been countless opportunities working with him, both in terms of the several collaborative projects and writing opportunities with I’ve enjoyed, as well as a diverse set of techniques taught to broaden my skillset. His advising style has always been favorable to me, hands-off when I wanted to work out a question to my own satisfaction, but also always available for advice and willing to work side-by-side when I asked. My presentation abilities were a particular weak point for me at the beginning, but regular practice with him and his willingness to push and challenge me proved to be exactly the help I needed. Many that have worked in the lab have helped me out, and have been great to have around. In particular, I’ve been lucky to have had lab partners like Daniella Sandoval, Rebeca Ibarra, and Connor Hill. Daniella and Connor both expressed countless milligrams of recombinant protein for my use, and Rebeca, in addition to the substantial amount of work she has put into tissue culture experiments towards my final paper, has also been an expert of organization and someone I could always ask when I couldn’t find something. Amy Ziemba also set me on the right path more than a couple times when I was a clueless undergraduate just getting started in the lab. My graduate committee, comprised of Drs. Hui Zhang, Ernesto Abel-Santos, and Laurel Raftery, was always helpful and kind in their guidance. They’ve offered strong advice during my regular meetings, and were always available outside of scheduled meetings. I am very thankful to have them serve on my committee. v The research I’ve performed is inextricably linked to collaborations, both local and distant. At UNLV, Lorena Samentar generated hundreds of confocal images for us, and Casey Hall in the Genomics Core has probably sequenced at least a thousand DNA samples for me personally. Their contributions are highly appreciated and their friendly demeanor always welcome. Outside of UNLV, I am indebted to Drs. Joe Harrison, Brian Kuhlman, Kurt Reichermeier, Brenda Schulman, Danny Scott, Frank Sicheri, and Mike Tyers, for the opportunities they gave our lab to work with them. None of my papers would have been the same without them if not for their uncanny ability to offer valuable reagents and priceless data just when I needed it the most. Everyone I’ve worked with, locally or thousands of miles away, has given me new appreciation for the value of cooperation in science. Some of the work within the grant was funded in part by the NIH grants R15 GM117555-01 and P20 GM103440. My family is the most important of all to me, and I am very thankful to have had them at my back my entire life. My parents, Brent and Caroline, have always supported me in every way possible, and allowed me to learn and study at my own pace, which was a vital skill to gain before setting out to obtain a graduate degree. My brothers, Parker, Mason, and Connor are my best buddies and also inspirational for their own respective pursuits that drive them in life. I’d also like to extend my thanks to my grandparents. Wayne and Howard were the first chemists of the family, and certainly sparked a love for chemistry in me at a young age, and Donna and Edna have been encouraging and supportive of my studies as well. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................................. iii ACKNOWLEDGEMENTS .......................................................................................................... v TABLE OF CONTENTS ............................................................................................................ vii LIST OF TABLES ........................................................................................................................ x LIST OF FIGURES ..................................................................................................................... xi CHAPTER 1 INTRODUCTION .................................................................................................. 1 1.1 The early history of the ubiquitin system ............................................................................... 1 1.2 Background
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  • Recombinant Human NEDD8 E1/APPBP1/UBA3 Protein Catalog Number: ATGP1425

    Recombinant Human NEDD8 E1/APPBP1/UBA3 Protein Catalog Number: ATGP1425

    Recombinant human NEDD8 E1/APPBP1/UBA3 protein Catalog Number: ATGP1425 PRODUCT INPORMATION Expression system E.coli Domain 1-463aa UniProt No. Q8TBC4 NCBI Accession No. AAH22853 Alternative Names NEDD8-activating enzyme E1 catalytic subunit, uBE1C PRODUCT SPECIFICATION Molecular Weight 54.4 kDa (487aa) Concentration 1mg/ml (determined by Bradford assay) Formulation Liquid in. 20mM Tris-HCl buffer (pH 8.0) containing 20% glycerol, 1mM DTT Purity > 90% by SDS-PAGE Tag His-Tag Application SDS-PAGE Storage Condition Can be stored at +2C to +8C for 1 week. For long term storage, aliquot and store at -20C to -80C. Avoid repeated freezing and thawing cycles. BACKGROUND Description uBA3, also known as NEDD8-activating enzyme E1 catalytic subunit, is the catalytic subunit of the dimeric uBA3- NAE1 E1 enzyme. E1 activates NEDD8 by first adenylating its C-terminal glycine residue with ATP, thereafter linking this residue to the side chain of the catalytic cysteine, yielding a NEDD8-uBA3 thioester and free AMP. E1 finally transfers NEDD8 to the catalytic cysteine of uBE2M. Recombinant human uBA3 protein, fused to His-tag at N-terminus, was expressed in E. coli and purified by using conventional chromatography. 1 Recombinant human NEDD8 E1/APPBP1/UBA3 protein Catalog Number: ATGP1425 Amino acid Sequence MGSSHHHHHH SSGLVPRGSH MGSHMADGEE PERKRRRIEE LLAEKMAVDG GCGDTGDWEG RWNHVKKFLE RSGPFTHPDF EPSTESLQFL LDTCKVLVIG AGGLGCELLK NLALSGFRQI HVIDMDTIDV SNLNRQFLFR PKDIGRPKAE VAAEFLNDRV PNCNVVPHFN KIQDFNDTFY RQFHIIVCGL DSIIARRWIN GMLISLLNYE DGVLDPSSIV PLIDGGTEGF KGNARVILPG MTACIECTLE LYPPQVNFPM CTIASMPRLP EHCIEYVRML QWPKEQPFGE GVPLDGDDPE HIQWIFQKSL ERASQYNIRG VTYRLTQGVV KRIIPAVAST NAVIAAVCAT EVFKIATSAY IPLNNYLVFN DVDGLYTYTF EAERKENCPA CSQLPQNIQF SPSAKLQEVL DYLTNSASLQ MKSPAITATL EGKNRTLYLQ SVTSIEERTR PNLSKTLKEL GLVDGQELAV ADVTTPQTVL FKLHFTS General References Gong L., et al.