1. BioSci D145 Lecture #9 Bruce Blumberg (blumberg@uci.edu) –4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) –phone 824-8573 TA – Ron Leavitt (rleavitt@uci.edu) –4351 Nat Sci 2, 824-6873 – office hours M 2:30-3:30 4206 Nat Sci 2 Updated lectures will be posted on web pages after lecture –http://blumberg.bio.uci.edu/biod145-w2016http://blumberg.bio.uci.edu/biod145-w2016 –http://blumberg-lab.bio.uci.edu/biod145-w2016http://blumberg-lab.bio.uci.edu/biod145-w2016 Term papers due Friday, March 4 by 12 midnight (23:59.59) (-1 point for each day late) Last year’s final exam is posted BioSci D145 lecture 9 page 1 © copyright Bruce Blumberg 2006-2016. All rights reserved

2. BioSci D145 lecture 9 page 2 © copyright Bruce Blumberg 2006-2016 All rights reserved Phenotypic rescue by RNAi – synthetic lethal and related approaches How can we find other members of pathways we already know something about? –Or, how can we find drugs that act on a pathway to kill cells (e.g., cancer cells?) –Synthetic lethal is one relatively new and promising approach 2 mutations are synthetic lethal if either single mutation is viable but the double mutant is lethal

3. BioSci D145 lecture 9 page 3 © copyright Bruce Blumberg 2006-2016 All rights reserved Phenotypic rescue by RNAi – synthetic lethal and related approaches How can we find other members of pathways we already know something about? –In cancer screening, what if we combine a mutation and a drug to find a combination that increases the kill rate, or reveals a phenotype that is similar to the total loss of function? Could find novel drug targets (pathways that kill cells in presence of siRNA+drug (usually sublethal amount of drug) Can find genes that are targeted by drugs – pathway analysis Could find new biomarkers that are required for cell viability following drug treatment

4. BioSci D145 lecture 9 page 4 © copyright Bruce Blumberg 2006-2016 All rights reserved Mohr et al., 2010, Genomic Screening with RNAi: Results and Challenges. Annual Review of Biochemistry, 29: 37-64

5. BioSci D145 lecture 9 page 5 © copyright Bruce Blumberg 2006-2016 All rights reserved Genome wide analysis of gene function How to mutate all genes (we know about) in a given genome? –Easy with microbial genomes – can mutate all yeast genes by homologous recombination –Recombine in selectable marker –Propagate strain and analyze phenotypes Target gene Selectable marker (antibiotic resistance) Homology region Unique oligonucleotide “barcodes” for PCR

6. BioSci D145 lecture 9 page 6 © copyright Bruce Blumberg 2006-2016 All rights reserved Genome wide analysis of gene function (contd) How about gene targeting in other organisms –With more complex genomes and more genes? –Huge undertaking to specifically target 30K+ genes in mammalian cells Difficulty Expense Inability to target all possible loci –Some efforts to make mouse collection Lexicon Genetics has a collection of ES cells –Drosophila collection as well –Driving force behind these efforts is Genome annotation Drug target discovery (Lexicon) Functional analysis

7. BioSci D145 lecture 9 page 7 © copyright Bruce Blumberg 2006-2016 All rights reserved Figure 5.18 The three major types of mutagen

8. BioSci D145 lecture 9 page 8 © copyright Bruce Blumberg 2006-2016. All rights reserved Genome wide analysis of gene function (contd) A main method for gene targeting in more complex organisms is random insertional mutagenesis –Transposon mutagenesis Bacteria – Tn transposons Yeast - Ty transposons Drosophila - P- elements Vertebrates - Sleeping Beauty transposons –Viral infection Typically retroviruses – host range selectivity is obstacle –Gene or enhancer trapping – modified viruses or transposons

9. BioSci D145 lecture 9 page 9 © copyright Bruce Blumberg 2006-2016. All rights reserved Insertional mutagenesis - enhancer trip enhancer trap is designed to bring inserted reporter gene under the control of local regulatory sequences –put a reporter gene adjacent to a weak promoter (enhancer-less), e.g. a retrovirus with enhancers removed from the LTRs –may or may not disrupt expression –Hopkins zebrafish group used unmodified virus viruses and transposable elements can deliver DNA to random locations –can disrupt gene function –put inserted gene under the control of adjacent regulatory sequences –BOTH

10. BioSci D145 lecture 9 page 10 © copyright Bruce Blumberg 2006-2016. All rights reserved Insertional mutagenesis - enhancer trap (contd) Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Nagayoshi S, Hayashi E, Abe G, Osato N, Asakawa K, Urasaki A, Horikawa K, Ikeo K, Takeda H, Kawakami K. Development 2008 Jan;135(1):159-69.

11. BioSci D145 lecture 9 page 11 © copyright Bruce Blumberg 2006-2016. All rights reserved Insertional mutagenesis - enhancer trap enhancer trap (contd) –expression only when integrate into an active transcription unit reporter expression duplicates the temporal and spatial pattern of the endogenous gene –reporters used  -galalactosidase was the most widely used reporter GFP is now popular  -lactamase is seeing increasing use –advantages relatively simple to perform active promoters frequently targeted, perhaps due to open chromatin –Disadvantages Inactive promoters probably not targeted insertional mutagenesis not the goal, and not frequent –overall frequency is not that high relies on transposon or retroviruses to get insertion –may not be available for all systems, requires transgenesis or good viral vectors

12. BioSci D145 lecture 9 page 12 © copyright Bruce Blumberg 2006-2016. All rights reserved expressed gene trap (many variations possible) –goal -> ablate expression of endogenous gene, replace with transgene –Make insertion construct – reporter, selection, polyA sites No promoter but has a splice-acceptor sequence 5’ of reporter Can only be expressed if spliced into an endogenous mRNA –Transfer into embryonic cells, generate a library of insertional mutagens Mouse, Drospophila, zebrafish, frog –reporter expression duplicates the temporal and spatial pattern of the endogenous gene Insertional mutagenesis – Gene trapping

13. BioSci D145 lecture 9 page 13 © copyright Bruce Blumberg 2006-2016. All rights reserved Insertional mutagenesis - Gene trapping (contd) Expressed gene trapping (contd) –advantages insertional mutagen –gives information about expression patterns –can be made homozygous to generate phenotypes higher efficiency than original trapping methods selectable markers allow identification of mutants –many fewer to screen –dual selection strategies possible –disadvantages overall frequency is still not that high frequency of integration into transcription unit is not high either relies on transposon or retroviruses to get insertion –may not be available in your favorite system. –Uses Insertional mutagenesis Marking genes to identify interesting ones Gene cloning http://www.genetrap.org/

14. BioSci D145 lecture 9 page 14 © copyright Bruce Blumberg 2006-2016.. All rights reserved Antisense methods to inhibit gene function Antisense oligonucleotides can transiently target endogenous RNAs –For destruction Many methods and oligo chemistries available Most are very sensitive to level of antisense oligo, these are degraded and rapidly muck up cellular nucleotide pools leading to toxicity –For translational inhibition Morpholino oligos appear to work the best –Morpholine sugar is substituted for deoxyribose –Is not a substrate for cellular DNAses or RNAse H –Base-pairs with RNA or DNA more avidly than standard DNA –The oligo binds to the area near the AUG in the transcript and inhibits translation of the protein – Deoxyribose morpholine

15. BioSci D145 lecture 9 page 15 © copyright Bruce Blumberg 2006-2016.. All rights reserved OligodeoxyribonucleotideMorpholino Oligonucleotide B = A, C, T, G Antisense methods to knock out gene function (contd)

16. BioSci D145 lecture 9 page 16 © copyright Bruce Blumberg 2006-2016.. All rights reserved Antisense methods to knock out gene function Morpholinos (contd) –For translational inhibition AUG morpholinos – make within about 50 bp of AUG –Inhibits translation of the mRNA but mRNA is still present

17. BioSci D145 lecture 9 page 17 © copyright Bruce Blumberg 2006-2016.. All rights reserved Antisense methods to knock out gene function Morpholinos (contd) –For translational inhibition Splice morpholinos are very effective –Target intron exon borders with the morpholino –Morpholino prevents splicing –No splicing -> no mature mRNA -> no transport out of nucleus –Or mis-splicing to get nonsense proteins »Or to get some unexpected product…. –Mature mRNA is depleted from cells leading to loss of protein

18. BioSci D145 lecture 9 page 18 © copyright Bruce Blumberg 2006-2016.. All rights reserved Antisense methods to knock out gene function Morpholinos (contd) –AUG morpholinos – make within about 50 bp of AUG Inhibits translation of the mRNA but mRNA is still present –Splice morpholinos are very effective Block or alter splicing to make no, or non-functional How do we verify that morpholinos worked as expected? –AUG morpholinos Western blot to verify loss of protein – requires an antibody Rescue with mRNA to which MO doesn’t bind –Most frequently used method in Xenopus Obtain same phenotype with a different MO −Good but gets EXPENSIVE –splice morpholinos RT-PCR to test for mature mRNA QPCR to quantitate

19. BioSci D145 lecture 9 page 19 © copyright Bruce Blumberg 2006-2016.. All rights reserved Most Molecules Function in Complexes Given a target, how can we identify interacting proteins? Complex members may be important new targets –pharmacology –toxicology –Endocrine disrupter action High throughput, genome wide screen is preferred –20 years is too long

20. BioSci D145 lecture 9 page 20 © copyright Bruce Blumberg 2006-2016.. All rights reserved How can we approach whole genome analysis of protein complex formation? Each protein interacts with average of 3 others Many are much more complex Papers this Thursday describe two different approaches to this problem.

21. BioSci D145 lecture 9 page 21 © copyright Bruce Blumberg 2006-2016.. All rights reserved How to identify protein-protein interactions on a genome wide scale? You have one protein and want to identify proteins that interact with it You want to identify all proteins that interact with all other proteins –straight biochemistry Co-immunoprecipitation GST-pulldown –Library based methods phage display Yeast two hybrid in vitro expression cloning –Proteomic analysis –Protein microarrays –Large scale two-hybrid

22. BioSci D145 lecture 9 page 22 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions biochemical approach – –what are some ways to purify cellular proteins that interact with your protein –pure protein(s) are microsequenced –advantage functional approach stringency can be manipulated can identify multimeric proteins or complexes will work if you can purify proteins –disadvantages much skill required low throughput considerable optimization required co-immunoprecipitation GST-pulldown affinity chromatography biochemical fractionation

23. BioSci D145 lecture 9 page 23 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) GST (glutathione-S- transferase) pulldown assay –Versatile and general –Fuse protein of interest to GST –Incubate with cell or tissue extracts –Mix with glutathione- sepharose beads Binds GST-fusion protein and anything bound to it –Run SDS-PAGE –Identify bands Co-IP (immunoprecipitation) is identical except that antibody is used to pull down protein X Many sorts of tags can be used

24. BioSci D145 lecture 9 page 24 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) scintillation proximity assay –Target is bound to solid phase – bead or plate –radioactive protein or ligand is added and allowed to reach equilibrium 35 S, 125 I, 3 H work best –radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target –applications ligand-receptor binding with 3 H small molecules protein:protein interaction protein:DNA

25. BioSci D145 lecture 9 page 25 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) FRET - fluorescent resonance energy transfer –transfer of energy from one fluor to another not normally excited at that wavelength –Many types of fluorescent moieties possible rare earth metals –europium cryptate fluorescent proteins –GFP and variants –allophycocyanin Tryptophan residues in proteins Use in protein:protein interactions? −If proteins are close AND if emission of A matches excitation of B FRET occurs

26. BioSci D145 lecture 9 page 26 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) FRET (contd) –application commonly used for protein:protein interaction screening in industry FRET microscopy can be used to prove interactions between proteins within single cells –Roger Tsien at UCSD is expert –advantages can be very sensitive may be inexpensive or not depending on materials non-radioactive equilibrium assay single cell protein:protein interactions possible time resolved assays possible –disadvantage poor dynamic range - 2-3 fold difference full scale must prepare labeled proteins or ligands –Difficult to do whole genome analysis this way multiwavelength capable fluorometer required (we have one here)

27. BioSci D145 lecture 9 page 27 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) Biacore (surface plasmon resonance) –surface plasmon waves are excited at a metal/liquid interface –Target bound to a thin metal foil and test sample flowed across it –Foil is blasted by a laser from behind SPR alters reflected light intensity at a specific angle and wavelength Binding to target alters refractive index which is detected as change in SPR Change is proportional to change in mass and independent of composition of binding agent

28. BioSci D145 lecture 9 page 28 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) Biacore (contd) –Advantages Can use any target Biological extracts possible Can measure kinetics –Generate K d directly Small changes detectable with correct instrument –360 d ligand binding to 150 kd antibody Can use as purification and identification system –Disadvantages Machine is expensive (we have three) “high throughput” very expensive Not trivial to optimize

29. BioSci D145 lecture 9 page 29 © copyright Bruce Blumberg 2006-2016.. All rights reserved Library-based methods to map protein-protein interactions (contd) Phage display screening (a.k.a. panning) –requires a library that expresses inserts as fusion proteins with a phage capsid protein most are M13 based some lambda phages used What is wrong with this picture? –prepare target protein as affinity matrix or as radiolabeled probe –test for interaction with library members if using affinity matrix you purify phages from a mixture if labeling protein one plates fusion protein library and probes with the protein –called receptor panning based on similarity with panning for gold Lambda and M13 phages don’t have legs…

30. BioSci D145 lecture 9 page 30 © copyright Bruce Blumberg 2006-2016.. All rights reserved Library-based methods to map protein-protein interactions (contd) Phage display screening (a.k.a. panning) (contd) –advantages stringency can be manipulated if the affinity matrix approach works the cloning could go rapidly –Disadvantages Multiple attempts required to optimize binding Limited targets possible may not work for heterodimers unlikely to work for complexes panning can take many months for each screen –Greg Weiss in Chemistry is local expert

31. BioSci D145 lecture 9 page 31 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) Two hybrid screening –originally used in yeast, now other systems possible –prepare bait - target protein fused to DBD (GAL4) usual stable cell line is commonly used –prepare fusion protein library with an activation domain - prey –What is the key factor required for success? no activation domain in bait! –approach transfect library into cells and either select for survival or activation of reporter gene purify and characterize positive clones

32. BioSci D145 lecture 9 page 32 © copyright Bruce Blumberg 2006-2016.. All rights reserved Mapping protein-protein interactions (contd) (stopped here) Two-hybrid screening (contd) –Can be easily converted to genome wide searching by making haploid strains, each containing one candidate interactor –Mate these and check for growth or expression of reporter gene Bait plasmidPrey plasmid If interact, reporter expressed and/or Yeast survive

33. BioSci D145 lecture 9 page 33 © copyright Bruce Blumberg 2006-2016.. All rights reserved Large scale mapping of protein-protein interactions GST (glutathione-S-transferase) pulldown assay –Or other purification wherein one protein is tagged and complex of proteins binding to it is recovered –Purify complexes from cells –Characterize complexes by mass- spectrometry –Iteratively build up a map of protein interactions from such complexes

34. BioSci D145 lecture 9 page 34 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression Proteomics is name given to study of the proteome (what is proteome)? Methods –2-D gel electrophoresis –Mass spectrometry of various sorts All mass spectrometry requires that molecules “fly” and measures mass/charge (m/z) ratio MALDI-TOF –Matrix assisted laser desorption ionization – time of flight –Laser causes matrix to vaporize and molecules to fly, charge is applied and time molecule takes to fly to detector measured along with m/z ESI –electrospray ionization – molecules are sprayed, ionized and detected MS-MS –Tandem mass spec – has two mass analyzers - first detector shunts selected molecule to second – used for sequencing and structure analysis –Proteome -> a cell or organism’s complement of expressed proteins Not necessarily identical to transcriptome

35. BioSci D145 lecture 9 page 35 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) 2-D electrophoresis –Ironically, this is the oldest method for “proteomics” –First dimension is isoelectric focusing Set up a pH gradient in tube, apply proteins and electrophorese each protein migrates to its isoelectric point and stops –Second dimension is SDS-PAGE – proteins migrate according to size Run at 90º to first dimension

36. BioSci D145 lecture 10 page 36 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) 2-D electrophoresis –Current technology is to cut out spots and id by mass spec Mass spec resurrected 2-D electrophoresis – Steep pH gradient shallow pH gradient

37. BioSci D145 lecture 10 page 37 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) 2-D electrophoresis (contd) –Good points Straightforward separation Can get good resolution with good isoelectric focusing gels –Downside Protein may not be detectable as well-resolved spots that can be excised and characterized –Co-migrate –Abundance Variation from experiment to experiment –Spot position on gel is very sensitive to small changes in pH

38. BioSci D145 lecture 10 page 38 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) Mass spectrometric methods –MudPIT is most useful for large scale protein profiling Multidimensional protein identification technology –Separate proteins by microcapillary liquid chromatography –Characterize and identify proteins by ms-ms –Prof. Lan Huang is local expert on protein profiling by mass spectrometry https://webfiles.uci.edu/lanhuang/www/

39. BioSci D145 lecture 10 page 39 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) Strategies for high-throughput, high-resolution protein identification and analysis –Equipment is very expensive but possibilities are limitless –Can match proteins with database sequences OR –Can sequence proteins de novo Computationally intensive

40. BioSci D145 lecture 9 page 40 © copyright Bruce Blumberg 2006-2016.. All rights reserved Global profiling of protein expression (contd) Protein arrays now available –Immobilized proteins Spot proteins on slides and ask what interacts with particular ones Luis Villareal runs a facility here that intends to produce all possible proteins for array generation (from Franciscella tularensis) –Antibody arrays Antibodies spotted on arrays – test for presence of particular proteins in probe Micro-ELISA or RIA –Antigen arrays Known antigens spotted – tests for presence of antibodies in sample