Department of Biochemistry
School of Medicine & Biomedical Sciences
BCH 405 Research Topics in Biochemistry

3 Credits, Fall Semester 2008
Course Coordinator: Dr. Murray Ettinger (829-3257; ettingem@buffalo.edu)

Wed. & Fri., 2:00-3:30 p.m., Farber 136

BCH 405 Course Description & Schedule 2008 [PDF]

Jump To:   SECTION 1    SECTION 2    SECTION 3    SECTION 4    SECTION 5  

 

FALL 2008 LECTURES

SECTION 1 - August 27, 2008 - September 12, 2008

 

Mechanisms of Eukaryotic RNA Polymerase II Transcription

Dr. Fred Ponticelli

Office: 649 Biomedical Research Building

Phone: 829-2473; email: asp@buffalo.edu

Class Schedule; Topics and Suggested / Required Reading: All information for Dr. Ponticelli's section is located on the UBLearns website.

 

SECTION 2 - September 17, 2008 - October 1, 2008

 

 

Dr. Gabriela Popescu

Office: 647 Biomedical Research Bldg.

Phone: 829-3807; email: popescu@buffalo.edu

SECTION 3 - October 3, 2008 - October 22, 2008

 

 

Dr. Murray Ettinger

Office: 647 Biomedical Research Bldg.

Phone: 829-3257; email: ettingem@buffalo.edu

Class Schedule; Topics and Suggested / Required Reading:  Printable copy - [PDF]  **Obtain a color copy of material where indicated**

The Role of Histone Methylation in Regulation of Gene Expression.

This subject illustrates rapid advances in a subject from initial discovery to molecular mechanisms to regulate gene expression to significance in regulation of cell proliferation and development and cancer.

1. Friday, October 3

INTRODUCTION
• Protein Methylation: What was known? What was unknown?
• Regulation of Gene Expression at the Level of Chromatin
• Histone Code Hypothesis

BACKGROUND TO PAPER 1
• Gene Silencing
• Vocabulary

2. Wednesday, October 8

The Discovery of SET-Domain Methyltransferases.
• How was this discovery made?

Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B., Sun, Z-W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C., and Jenuwein, T. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-599. [PDF] (*Color copy*)
Direct Link: http://www.nature.com/nature/journal/v406/n6796/pdf/406593a0.pdf

3. Friday, October 10

Based on the histone code hypothesis, what is the predicted effect of methylating a specific lysine in histone H3; i.e. what happens next?

Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., and Jenuwein, T. (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120. [PDF] (*Color copy*)
Direct Link: http://www.nature.com/nature/journal/v410/n6824/pdf/410116a0.pdf

4. Wednesday, October 15

How can methylation of a Lysine on a Histone create a specific binding site for a regulatory protein; i.e. how do methylated lysines bind to proteins, AND how is specificity determined (dictated) by the histone code?

Jacobs, S.A. and Khorasanizadeh, S. (2002) Structure of HP1 Chromodomain Bound to a Lysine-9-Methylated Histone H3 Tail. Science 295, 2080-2083.
[PDF] (*Color copy*)
Direct Link: (http://www.sciencemag.org/cgi/content/full/295/5562/2080)

5. Friday, October 17

The plot thickens: Are there other SET-Domain methyltransferases? Does methylation of specific Histone Lysines always lead to gene silencing?

Santos-Rosa, H., Schneider, R., Bannister, A., Sherriff, J., Bernstein, B., Emre, N., Schreiber, S., Mellor, J. and Kovzarides, T. (2002) Active genes are tri-methylated at K4 of histone H3. Nature 419, 407-411. [PDF]
Direct Link: http://www.nature.com/nature/journal/v419/n6905/pdf/nature01080.pdf

6. Wednesday, October 22

The term, "SET"-domain came from three proteins that were known to be involved in regulating development in Drosophila. "S" = SU-MTase that we read about. This class will be about the "E"-protein of "SET". Is it a MTase? What does it do? Significance? : development and cell proliferation (cancer)!

Müller, J., Hart, C., Francis, N., Vargas, M., Sengupta, A., Wild, B., Miller, E., O'Connor, M., Kingston, R., and Simon (2002) Histone Methyltransferase Activity of a Drosophila Polycomb Group Repressor Complex. Cell 111, 197-208.
[PDF] (*Color copy*)
Direct Link: (http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WSN-473VYYH-9-F&_cdi=7051&_orig=search&_coverDate=10%2F18%2F2002&_qd=1&_sk=998889997&view=c&wchp=dGLbVlz-zSkWb&_acct=C000037419&_version=1&_userid=681891&md5=bb0a8186dae3520cb771b8a709a8b74d&ie=f.pdf)

SECTION 4 - October 24, 2008 - November 7, 2008

Dr. Michael Buck

Office: B3-304 Center of Excellence in Bioinformatics Bldg., 701 Ellicott St.
Phone: 881-7569; email: mjbuck@buffalo.edu

SECTION 5 - November 12, 2008 - December 5, 2008

Dr. Kenneth Blumenthal
Office: 140 Farber
Phone: 829-2727; email: kblumen@buffalo.edu

Dr. Blumenthal - Powerpoint Presentation [PPT]

References

Class Schedule; Topics and Suggested / Required Reading  (word doc)

Protein-Protein Interactions and Cell Signaling Pathways

In these sessions, we will review biochemical and other methods for analyzing protein-protein interactions in the context of elucidating signal transduction pathways used by cells. In addition, we will introduce mass spectrometry as a new technology for high sensitivity, high resolution analysis of (a) the components of signaling complexes and (b) changes in cell protein compositions (termed "the proteome") caused by receipt of signals from other cells and/or the environment. Finally, we will discuss 1-2 papers in which mass spectrometry is the key tool for experiements involving either gene discovery or proteome alteration caused by either gene discovery or proteome alteration caused by either disease or bacterial infection. Note that the references given below for the two lectures may carry over into the third as well, and that we may not begin our discussion of the ras pathway until our third meeting. It would be very useful for students to read the minireviews of ras signaling prior to our discussing it in class.

Wednesday, November 12
Lecture 1: Discussion of Protein-Protein Interactions Methods
Affinity based (pulldowns of all sorts; see notes from ASP section)
   1. GST and other pulldowns
   2. Co-immunoprecipitation
   3. Cross-linking
   4. Y2H (yest 2-hybrid analysis)
Protein Electrophoresis
   1. Non-denaturing (native) polyacrylamide gels
   2. Denaturing (SDS) polyacrylamide gels
   3. Two-dimensional gels (isoelectric focusing combined with SDS-PAGE)
   2D-gels [1. Ref: Gygi et al (2000) "Evaluation of 2-D gel electrophoresis based proteome analysis technology," PNAS 97,    9390 (PDF)]
   4. Immunoblotting (aka, Western blotting)
Mass spec analysis [see PPT file; also Griffin and Aebersold (2001) "Advances in proteome analysis by mass spectrometry" J. Biol. Chem. 276, 45497 (PDF)]
   1. Principles and practice of mass spec.
   2. Protein fingerprinting by mass spec.
   3. Protein sequencing by mass spec.
   4. Database searching using mass spec data.

Friday, November 14
Lecture 2: Analysis of Cell Signaling Pathways
Refs:  Shevchenko, A., et al (2002) "Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry," Mol. Cell. Proteomics 1, 204-12 (PDF)
    1. Biochemical approaches
    2. Identification via pharmacology
    3. Identification via genetics
    4. Refs: cont'd as from above set.
    5. Introduction to ras, via GPCR-mediated signaling. Refs: review Matthews and Van Holde, Chapter 23, pp 864-868 describing ras and oncogenes; Satoh et al (1992) "Function of ras as a molecular switch in signal transduction," J. Biol. Chem. 267, 24149 (PDF); Schlessinger, J. (1993) "How receptor tyrosine kinases activate Ras," TIBS 18, 273-5

Wednesday, November 19 & Friday, November 21
Lecture 3/Lecture 4: Dissecting Signal Transduction Pathways: the ras Paradigm
   1. Molecular events regulating the cell cycle.
   2. Role of protein phosphorylation in cell cycle regulation.
   3. Role of protein complexes in cell cycle regulation.
   4. Importance of inter-pathway communication.
   5. Parallelism in the ras (and others) pathway.

Wednesday November 26th-Friday November 28th: Fall Recess No Classes

Wednesday, December 3 & Friday, December 5
Lecture 5/Lecture 6: PDGF Signaling Complexes:
More adventures with ras; discussion of paper: Vukic Soskic, Matthias Gorlach, Slobodan Poznanovic, Frank D. Boehmer, and Jasminka Godovac-Zimmermann (1999) “Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor receptor,” Biochemistry 38, 1757-1764 (PDF)

alt: Orkin from 2006 Nature (PDF)

Each student will be required to write a short paper based on the Nature review by Aebersold and Mann (PDF). This will be due on December 5th in electronic format. This paper should expand on a single topic in the review article. Examples of appropriate topics would include, but are not limited to, a description of essential designconsiderations in proteomics experiments, how proteomics can be applied in protein profiling, analysis of post-translational modifications, etc. Other appropriate subjects would be a description of areas (mostly technical) which need further development in order for proteomics to realize its full potential. This would necessarily require some analysis of what we can do now and where currently available approaches fall short. Papers should be 2-4 pages in length, and may includeas many figures as you wish. You should feel free to consult as many references (from the Nature paper, or elsewhere) as you wish in preparing this paper, but the paper must be in your own words, and all references must be cited.