Interactions of SUMO within the JAK/STAT transcription pathway A new method to analyze the regulation of the JAK/STAT immune response pathway using protein.

Slides:

Advertisements

Similar presentations

Step 1: Enzyme digest to remove stop codon from original plasmid Step 2: Mutagenesis PCR to remove stop codon Step 3: Mutagenesis PCR to remove 1071 G.

Advertisements

Analyzing the effects of a gene-specific siRNA on IL13R-α2 mRNA and protein levels in glioblastoma multiforme cells INTRODUCTION  RNA interference (RNAi)

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription.

Genetic Incorporation of Unnatural Amino Acids into Proteins Monica Amin Yang Song Yan Liu Harbani Malik Vipul Madahar Jiayu Liao Department of Chemical.

Cellular Communication. Chemical messages which elicit a response in cells serve as a form of communication between cells Found in all cells Similar in.

Basics of Molecular Biology

Developing a protein-interactions ontology Esther Ratsch European Media Laboratory.

BRIAN PHAN DR. JANE ISHMAEL DEPARTMENT OF PHARMACEUTICAL SCIENCES SUMMER 2010 Characterization of LC8 Interaction with NR1 subunit of NMDA Receptors.

Development of High-throughput Screening Assay for Small Molecule Inhibitor(s) of PIAS1 Bourns College of Engineering Department of Bioengineering Vicente.

Gene Expression Overview

Can Expression of VACM-1 Enhance Effects of Thalidomide on Endothelial Cell Growth? Drake Harper Zeeland East High School John Pelton Maria Burnatowska-Hledin.

Fluorescent Resonance Energy Transfer (FRET)-based Method for Dissecting Protein - Protein Interactions in Sumoylation Pathway Rachel Rattner Dr. Jiayu.

Signaling and the Signal Transduction Cascade. Question?????? External Stimulus Inside cell Nucleus, Gene transcription Other cellular effects.

Modeling the Interferon Signaling Process of the Immune Response Jeffrey Suhalim Dr. Jiayu Liao and Dr. V. G. J. Rodgers BRITE.

Genetic Incorporation of Unnatural Amino Acids into Proteins Monica Amin 1, Yang Song 2, Yan Liu 2, Harbani Malik 2, Vipul Madahar 2, Jiayu Liao 2 Department.

Cell Communication III. G-protein linked receptor.

Colinearity of Gene and Protein DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription translation.

IGF in circulation The majority (> 75 %) exists as bound form –IGF binding proteins (IGFBPs) IGFBPs –6 proteins and several related proteins –Serum IGFBP.

PROTEIN SYNTHESIS BY: MARIAH GUMFORY. OBJECTIVES Explain the purpose and process of transcription and translation Recognize that gene expression is a.

Books Molecular Cell Biology Lodish

AP Biology Control of Eukaryotic Genes.

Synthetic biology: New engineering rules for emerging discipline Andrianantoandro E; Basu S; Karig D K; Weiss R. Molecular Systems Biology 2006.

Protein Synthesis: Ch 17 From : Kevin Brown – University of Florida

The initial RNA transcript is spliced into mature mRNA

Protein Synthesis How to code for the correct amino acids.

Centra Dogma Primer. Structure of DNA and RNA Nucleic acids made of nucleotides G, A, T/U, C Ribose vs. deoxyribose Template-dependent synthesis Double.

Molecular Basis for Relationship between Genotype and Phenotype DNA RNA protein genotype function organism phenotype DNA sequence amino acid sequence transcription.

Transcription control elements (DNA sequences) are binding sites for transcription factors, proteins that regulate transcription from an associated.

RESULTS EXPERIMENT CONCLUSION Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium Classes of Neurospora.

Chapter 12: The Cell Cycle

Agenda 07/20/2010 Sketch Transcription Quiz – genetics 1 Translation New Vocab Assembly Enzyme Synthesis Review Mutations – Research Genetic Engineering.

BIOCHEMISTRY REVIEW Overview of Biomolecules Chapter 13 Protein Synthesis.

Cell Communication Chapter Cell Communication: An Overview  Cells communicate with one another through Direct channels of communication Specific.

Control levels of transcription factor (TF) activity.

JAK-STAT Signaling Pathway.

Translation- Making the Protein

Protein Synthesis- the ”Stuff of Life“ Translation- Making the Protein.

Chapter 15- Cell Communication Part I- General signaling strategies

The Three Stages of Cell Signaling By: Madeline Meyer and Carlos Sanchez ReceptionTransductionResponse.

Protein Synthesis- the ”Stuff of Life“ Translation- Making the Protein.

Interferons Induction of synthesis Induction of antiviral activity Antiviral activities induced by interferons  and  Antiviral activities induced by.

Gene Regulation and Mutation Notes and Questions How do mutations affect a cell?

Agenda 3/9 Transcription Warm Up Translation Notes Protein Synthesis Model Homework: Chp 14 notes, Mutations WS, Protein Synthesis Video Turn in: Video.

Protein Synthesis. One Gene – One Enzyme Protein Synthesis.

Defining Epidermal Growth Factor Receptor exon 20 mutant sensitivity to tyrosine kinase inhibition Danny Rayes.

Protein Synthesis (Translation)

SERGIO J. DAVILA, David Jesse Sanchez, Genhong Cheng

Interferons: Type I José Ignacio Saldana, Imperial College London, UK

SIGNAL TRANSDUCTION Signal Transduction Pathway Protein Modification Phosphorylation Cascade Protein Kinases.

Gene Expression.

to photoreactive amino acid

Nat. Rev. Rheumatol. doi: /nrrheum

How DNA and RNA make Proteins.

Cell Signaling.

Figure 5 Defects in the JAK–STAT signalling pathway

Cell to Cell Communication via Enzyme Linked Receptors

β-ARs signal cooperatively with mutant EGFR and inactivate LKB1

Chapter 17 From gene to protein.

Lizabeth Allison Ch:14 Waever Ch 17, 18, 19

Step 2 of protein Synthesis

AP Biology The Cell Cycle.

Volume 15, Issue 5, Pages (November 2001)

RAD51 is essential for L. donovani.

Protein Synthesis.

Per Stehmeier, Stefan Muller Molecular Cell

credit: modification of work by NIH

Chapter 12: The Cell Cycle

Figure 2 Signalling downstream of the IL-6 receptor

Vps36 interacts with Smo in the absence of Hh

Volume 7, Issue 1, Pages 1-11 (July 1997)

Presentation transcript:

Interactions of SUMO within the JAK/STAT transcription pathway A new method to analyze the regulation of the JAK/STAT immune response pathway using protein engineering Adam Cheng Xiulin Shen Prof. Jiayu Liao Department of Bioengineering

Introduction ► 1918 flu pandemic ► JAK/STAT pathway ► SUMO regulation ► Goals and Strategies ► Methods ► Results ► Conclusion

Spanish Flu Pandemic of

JAK/STAT Pathway ► Cytokines like interferon send messages throughout the body

JAK/STAT Pathway ► JAK phosphorylates the receptor and STAT ► STAT dimer stimulates transcription

JAK/STAT Pathway ► Outcomes from the JAK/STAT1 Pathway:  Immunoglobulin G  Protein Kinase R (PKR)  TH1 Differentiation (T-Cells)

SUMO regulation ► SUMO protein cascade ► PIAS and SENP function ATP E1 SE2 S E3 (PIAS) STAT SENP SUMO STAT

Goal ► Investigate signaling specificity by analyzing SUMO’s interactions using a new strategy of incorporating unnatural amino acids ► Understand the regulation mechanisms of cytokine signaling pathways

Strategy ► Mutate essential interactive amino acids between SUMO and SENP to alanine so SENP cannot recognize SUMO ► Mutate neighboring amino acids to p- benzoyl-L-phenylalanine to permanently link and fish out interaction partners

Methods ► Mutations at/around these amino acids

Crosslinking unnatural amino acid: pBpa – p-benzoyl-L-phenylalanine

Wild Type Protein Synthesis ► Essential parts in protein synthesis: ss430/lecture%209-07/figure JPG idson.edu/courses/ genomics/2005/Dry sdale/molecular%2 0function.jpg

Incorporating pBpa in vivo ► What is needed for unnatural amino acid incorporation?  1) Unnatural amino acid (pBpa)  2) Engineered tRNA  3) Engineered Aminoacyl-tRNA synthetase

tRNA requirements ► Codon mutated to TAG (stop codon) ► Unrecognized by endogenous synthetases

Aminoacyl-tRNA Synthetase Requirements ► Only aminoacylates the engineered tRNA ► Only accepts unnatural amino acid into active site

Methods: Mutation ► Mutations through PCR primers/hybridization ► Restriction digestion of DNA ► Ligation to mammalian expression vector

Methods: Expression ► Transfection of STAT1/alanine SUMO1/SENP2 ► Lyse cells and extract protein

Methods: Expression ► Transfection of STAT1/pBpa SUMO1/SENP2/TAG tRNA/Aminoacyl-tRNA synthetase ► Add p-benzoyl-L-phenylalanine (1mM) 6h after transfection SUMO/SENP complex

Methods: Analysis ► Analysis using Western Blot SUMO STAT/SUMO SUMO/SENP big small Insert protein here

Results and Future Work ► All mutations are complete ► Analyze using Western Blot ► Affirm results using another method ► Move techniques to related pathways  Anti-tumor p53 pathway

Conclusion ► A new amino acid incorporation strategy was applied ► Two types of SUMO mutations were generated to further the study of the JAK/STAT pathway ► Results will benefit human health

Acknowledgements ► Yang Song ► Vicente Nuñez ► Vipul Madahar ► Clarence Pasion ► Xiulin Shen ► Department of Bioengineering ► Maria Franco-Aguilar and UCLEADS ► Dr. Victor Rodgers, Jill Brady, BRITE

Ackowledgements ► Dr. Jiayu Liao

References ► D. Schmidt and S. Miller. (2003) PIAS/SUMO: Partners in transcriptional regulation. Cell. Mol. Life. Sci. 60: ► Johnson, Erica S. (2004) Protein Modification by SUMO. Annu. Rev. Biochem. 73: