Damian Guerra, PhD.
105 Colonial Village Apt 105
Amherst, MA 01002
CURRICULUM VITAE
Damian Guerra
Postdoc, Laboratory of Elizabeth Vierling (beginning April 2013)
1003 Lederle Graduate Research Tower
Department of Biochemistry and Molecular Biology
University of Massachusetts, Amherst
710 N. Pleasant St.
Amherst, MA 01003
PHONE: 413-545-0469 (Lab) 208-596-9014 (Cell)
FAX: (413) 545-3291
Email: ddguerra@ucdavis.edu
SUMMARY OF QUALIFICATIONS
Biochemist specializing in protein purification, in vitro enzyme assays, antibody purification and immunoprecipitation, subcellular fractionation, DNA sequence manipulation and cloning, and the production of transgenic Arabidopsis to assess gene function. Experience with DNA and protein gel electrophoresis, recombinant protein expression in E. coli, Western blotting, immunoprecipitation, DNA isolation and purifications, PCR and standard recombinant DNA technologies. Motivated research scientist with classroom, laboratory, and private instructor teaching experience.
PROFESSIONAL PREPARATION
Lewis-Palmer High School, Monument, Colorado May 2004
Valedictorian
Washington State University, Pullman, Washington May 2007
Bachelor of Science, Summa cum laude
Major: Biochemistry and Molecular Biology
University of California, Davis, Davis, California March 2013
Ph.D.
Major: Biochemistry, Molecular, Cell and Developmental Biology
PUBLICATIONS
Pratelli R*, Guerra D*, Yu S, Wogulis M, Kraft E, Frommer WB, Callis J, Pilot G. (2012). The ubiquitin E3 ligase LOSS OF GDU 2 is required for GLUTAMINE DUMPER 1- induced amino acid secretion in Arabidopsis. Plant Physiol. 158(4):1628-42. (*: authors contributed equally to this work).
Guerra D and Callis J (2012) Ubiquitin on the move: the ubiquitin modification pathway plays diverse roles in the regulation of ER- and plasma membrane-localized proteins. Plant Physiol. 160(1): 56-64.
Guerra D*, Pratelli R*, Kraft E, Callis J, Pilot G. (2013). The Mammalian E3 ligase MAHOGUNIN RING FINGER 1 interacts with plant GLUTAMINE DUMPER 1 via the conserved DOMAIN ASSOCIATED WITH RING2 region. FEBS Lett. Submitted March 14th 2013.
Guerra D*, Pratelli R, Jia W, Leary J, Pilot G, and Callis J (2013). LOSS OF GDU2 regulates GLUTAMINE DUMPER 1 abundance and promotes the Gdu1D phenotype in a manner requiring its ubiquitin ligase activity in Arabidopsis thaliana. Manuscript in preparation.
INVITED PRESENTATIONS
Nara Institute of Science and Technology (NAIST), 4th International Graduate Student Workshop, Nara, Japan, Nov. 2010 (presented lecture).
NAIST 6th Global COE International Symposium, 2010: “Plasticity in Development and Evolution.” Nara, Japan, Nov. 2010 (presented poster).
UC Davis BMCDB fall colloquium, Davis, CA, 2011 (presented lecture).
MEETING ABSTRACTS
D. Guerra, R. Pratelli, S. Yu, M. Wogulis, E. Kraft, W. Frommer, G. Pilot, and J. Callis. (2012). “LOG2, a plasma membrane-anchored ubiquitin ligase, interacts and co-localizes with GDU1, a membrane protein involved in the control of amino acid export.” 4th Pan-American Workshop in Plant Membrane Biology, Pacific Grove, CA.
D. Guerra, R. Pratelli, M. Wogulis, E. Kraft, G. Pilot, and J. Callis. (2011). “Interactors of the Arabidopsis AKHERON ubiquitin E3 ligases implicate the ubiquitin pathway in amino acid homeostasis and stress response pathways.” Cold Spring Harbor annual meeting on the ubiquitin family, Cold Spring Harbor, NY.
D. Guerra, R. Pratelli, W. Wogulis, E. Kraft, G. Pilot, and J. Callis (2010). A family of RING-type ubiquitin ligases in Arabidopsis play a role in abiotic stress adaptation. NAIST 4th International Graduate Student Workshop, Nara, Japan.
KW. Kim, D. Guerra, L. Davin, and N. Lewis. (2007). “Towards defining the biochemical mechanism of stereoselective monolignol (radical) coupling with dirigent proteins: a site-directed mutagenesis approach.” Phytochemical Society of North America Annual Meeting, St. Louis, MO.
D. Guerra, F. Cochrane, D. Bedgar, L. Davin and N. Lewis. (2006). “Characterization of a putative bifunctional CCR/CAD in Arabidopsis thaliana.” Phytochemical Society of North America Annual Meeting, Oxford, MS.
AWARDS AND HONORS
Pan-American Workshop in Plant Membrane Biology Travel Award, 2012
Eric and Louise Conn Graduate Student Award in Plant Biochemistry, 2012
UC Davis College of Biological Sciences Work Study Award, 2011
National Institutes of Health Predoctoral Trainee, 2009-2011
Washington State University Center for Integrated Biotechnology fellowship, 2006
National Science Foundation Undergraduate research fellowship, 2005
PROFESSIONAL AFFILIATIONS
Phytochemical Society of North America
American Society of Plant Biologists
PROFESSIONAL APPOINTMENTS
Teaching assistant for undergraduate metabolic biochemistry (BIS 103, UC Davis) Spring 2009, Spring 2012.
Professor: Judy Callis, Ph. D.
Teaching assistants hold office hours, guide study sections, and grade exams.
Undergraduate Research Associate Aug 2004-Aug 2007
Research advisor: Norman Lewis, Ph. D.
Learned basic techniques in molecular biology and biochemistry including bacterial transformation, recombinant protein over-expression, protein purification via ion exchange column chromatography, enzyme assays to obtain Michaelis-Menten kinetic parameters, and the identification of small molecules via reverse phase HPLC.
PROFESSIONAL ACTIVITIES
Co-founder and organizer of UC Davis Biochemistry, Molecular, Cellular, and Developmental Biology Career Options Seminar Series (BMCDB COS), 2011-2012.
D. Melters, K. Garvey, and D. Guerra
While doctoral programs facilitate the training expected of a person holding an advanced degree, few graduate students consider career options outside the traditional academic postdoc trajectory. A seminar series was therefore conceived in which recent doctoral graduates pursuing non-traditional careers (i.e., freelance science journalism, science policy formation, dual academic and industrial appointments, etc.) explain how and why they chose their occupations. The BMCDB COS series has received positive reviews and is slated to be renewed next year.
Graduate student mentor to undergraduate researchers and junior specialists, 2009-2013.
UC Davis undergraduates and junior specialists are taught techniques applicable to basic research in biochemistry, molecular biology, and plant physiology.
The following current and former UCD undergraduate students have been trained:
Vincent Kam (2009-2010): B.S. (2010), Biochemistry and Molecular Biology. Current occupation: junior specialist in plant biology (Gina Coaker, UCD) (vkam@ucdavis.edu).
Kayla Benton (2011-): 4th year (B.S., 2012 expected), Cell Biology. (kmbenton@ucdavis.edu).
The following junior specialist has been trained:
Khin Kyaw (2010-): B.S. (2010, UC Davis), Biochemistry and Molecular Biology. (kwkyaw@ucdavis.edu).
Private tutor in graduate-level general biochemistry
Graduate students are tutored for 3-6 hours per week in the subject of general biochemistry (thermodynamics, protein folding, macromolecular structures, signal transduction pathways, techniques in molecular biology, and intermediary metabolism).
The following students have been tutored:
Michelle Ross (Feb-May 2010): M.S. (2011, U. Bridgeport), Human Nutrition. (michelle.ross@mac.com).
Teaching assistant for undergraduate metabolic biochemistry (BIS 103, UC Davis), 2009, 2012.
Teaching assistants hold office hours, guide study sections, and grade exams.
UC Davis BMCDB fall colloquium organizer, 2008.
The fall colloquium is organized by second-year BMCDB graduate students and is meant to introduce incoming students to the variety and depth of research at UC Davis. The event consists of lectures, catered lunch and dinner, a poster session, and extracurricular activities. Duties include fundraising, assembling a group of seminar speakers, planning extracurricular events, and infrastructure allocation.
Student host to speakers in the UC Davis molecular biology joint seminar series, 2010-2012
http://bioscinet.ucdavis.edu/Events/Events/JointSeminars
Graduate students invite distinguished professors to give research seminars and to meet with College of Biological Sciences faculty and students. The student host drafts the invitation, advertises the speaker’s visit, and arranges the speaker’s itinerary.
UC Davis BMCDB host for recruitment and orientation, 2009-2012
Recruitment and orientation hosts are responsible for producing itineraries, serving as a campus guides, and fostering discussion about the graduate program to new and prospective PhD students.
RESEARCH EXPERIENCE
University of California, Davis April 2008-March 2013
Ph.D. Student (Major advisor: Judy Callis, Ph.D.)
Determined the role of a ubiquitin E3 ligase in facilitating the amino acid export phenotype conferred by over-expression of a single-pass transmembrane protein in Arabidopsis. R. Pratelli*, D. Guerra*, et al. (2012). Plant Physiol. 158(4):1628-42. (*: authors contributed equally to this work).
Amino acids serve as transport forms for organic nitrogen in the plant and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, over-expression of GLUTAMINE DUMPER 1 (GDU1) in Arabidopsis thaliana led to increased amino acid export. To gain insight into GDU1’s role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS of GDU 2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. LOG2 has four paralogs in Arabidopsis (LOG2-LIKE UBIQUITIN LIGASE, or LUL1-4) and is homologous to the mammalian protein MAHOGUNIN RING FINGER 1. The interaction between LOG2 and GDU1 was confirmed by GST pull-down, in vitro ubiquitination, and in planta co-immunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant, log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial miRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.
Biochemical characterization of IXION, a novel protein that interacts with LOG2
D. Guerra, E. Kraft, K. Kyaw, and J. Callis. Manuscript in preparation.
LOG2 is now known to affect amino acid homeostasis. To uncover additional physiological functions of this gene family, a yeast-two-hybrid screen was conducted using LUL2 as bait. Among potential LUL2-interacting proteins was IXION, an uncharacterized polypeptide lacking annotated domains that is conserved in plants. LUL2-4 and LOG2 polyubiquitinated IXION in vitro, while both LOG2 and LUL3 directly interacted with IXION in GST pull-downs. Similarly to LOG2 and GDU1, IXION localized to endomembranes and was enriched in the plasma membrane in vivo. IXION protein levels prodigiously increased upon drought and heat stresses, although the additional stress of high light intensity blocked heat-induced protein accumulation. TDNA- and artificial microRNA-mediated knockdown of IXION marginally reduced organismal fitness upon the aforementioned stress treatments, and IXION protein abundance was unaffected by inhibition of either protein synthesis or ubiquitin-mediated proteolysis. However, IXION was found to interact with a BAX INHIBITOR-1-LIKE (BXL) protein in a yeast-two-hybrid experiment. Furthermore, both IXION and BXL are transcriptionally activated by heat and drought stresses. These results suggest IXION is an abiotic stress adaptation factor in plants.
Continuing characterization of the relationship between LOG2 and GDU1.
D. Guerra, W. Jia, R. Pratelli, G. Pilot, J. Leary, and J. Callis. Manuscript in preparation.
GDU1 is an in vitro E3 ligase substrate of LOG2, and LOG2 is necessary for GDU1 function in vivo. It is therefore reasonable to hypothesize that GDU1 ubiquitination by LOG2 is necessary for its physiological activity. To examine this possibility, I am collaborating with a mass spectrometry lab to identify the GDU1 residues ubiquitinated by LOG2. In vitro, the cytosolic domain of GDU1 was found to be ubiquitinated at lysines 29, 86, 98, and 100. Two of these residues—K29 and K86—were also uncovered as ubiquitination targets in planta. Intriguingly, arginine replacement of K86, K98, and K100 led to an accumulation of GDU1 in a transient tobacco expression system. Moreover, GDU1-myc protein levels were substantially higher in crude lysates, total microsomes, and the plasma membrane fractions of Arabidopsis that express lower levels of LOG2 (i.e., plants homozygous for the log2-2 mutation). Preliminary results further indicate that LOG2-HA transgenes harboring mutations in the myristoylation motif or RING domain do not rescue the Gdu1D phenotype to the same extent as a wild-type LOG2-HA transgene in the GDU1-myc log2-2 background. This suggests GDU1 may require ubiquitination (and subsequent degradation) to carry out its biological activity, and that myristoylation is important for the in vivo activity of LOG2 (probably by affecting its localization).
Washington State University, Institute of Biological Chemistry 2004-2007
Undergraduate Research Associate (Research advisor: Norman Lewis, Ph.D.)
Examined the contribution of conserved amino acids to the stereospecificity of monolignol radical-radical coupling by Dirigent Protein from Schisandra chinensis.
Lignans are plant secondary metabolites synthesized from phenylalanine and tyrosine via the phenylpropanoid pathway and subsequent stereospecific oligomerization by dirigent proteins (DPs). Many lignans have been shown to possess antimicrobial and anticancer activities. However, the mechanism of DP-catalyzed stereospecific radical-radical coupling is not well understood. A postdoc in the lab performed site-directed mutagenesis to substitute conserved amino acids of DP from Schisandra. I then recombinantly expressed these proteins in insect cells and purified them via cation exchange chromatography. Mutant DPs were assayed for their ability to facilitate the stereospecific dimerization of monolignol radicals. Products were analyzed by comparing in-line UV spectra of HPLC-separated analytes to reference standards. It was found that mutating conserved aromatic amino acids favored the formation of a racemic product, while mutation of a single serine residue enhanced the optical purity of the product. The findings of this investigation could aid in the engineering of DPs that increase the yield of biomedically-relevant lignans.
CAREER GOALS
I want to practice basic or clinical research by using and building upon the technical methods I have learned in graduate school. Following completion of my dissertation research, I will pursue a postdoctoral fellowship in a biochemistry-related subfield. A major goal for the next 2-4 years will be to reinforce and diversify my skills as a bench scientist. I am open to projects in the context of human health and disease since I have worked mostly with plants up to this point. Molecular techniques tend to be applicable across species, and as I consider myself a biochemist first and a biologist second. I excel at teaching and may wish to serve as an educator (i.e., as a professor) later in my career, but I want to focus on research for the next 2-4 years. Whether I pursue a career in industry or apply for an academic faculty position will depend on my experiences as a postdoc.
RESEARCH PROJECT INTERESTS
Broadly, I am interested in signal transduction and intermediary metabolic pathways, and biochemical and physiological adaptive responses to stress. Changes in gene expression, protein longevity, and membrane composition that accompany the transition from normal to pathogenic states such as cancer are of particular interest.
PROFESSIONAL REFERENCES
Judy Callis (PhD Advisor)
Paul K. and Ruth R. Stumpf Chair in Plant Biochemistry
Professor of Molecular and Cellular Biology
University of California, Davis
Email: jcallis@ucdavis.edu
Phone: 530-752-1015
Diane Beckles
Assistant Professor of Plant Biochemistry
University of California, Davis
Email: dmbeckles@ucdavis.edu
Phone: 530-754-4779
Robert Fairclough
Associate Professor of Neurology
University of California, Davis
Email: rhfairclough@ucdavis.edu
Phone: 530-754-5005
Michael Smerdon
Regents Professor of Biochemistry and Biophysics
Washington State University
Email: smerdon@wsu.edu
Phone: 509-335-6853