IGEM:MIT/2005/PATHWAYS!!

Pathways We'll Try

• Put pathways you think is worth a try on here. Eventually, try to make a list of 5 or less.
• Criteria:

best is dimerization system, conformational change is considerable

has characterized signal path

not essential to the cells

Information

• Cell Membrane Pathways
• KEGG Pathway Database
• Links to Collections of Pathways
• Different Types of Cytokine Receptor
• BioCarta
• Statistics about E.Coli
• Kabat Database for Antibodies
• Ig BLAST

ToxR

• ToxR/lambda phage
  • Read the intro to this article (not just the abstract). Its VERY informative --> good for beginners.
• Co-regulation of cholera toxin genes
  • ToxR’s role is signal transduction through the membrane. It’s mechanism of action is thought to be the dimerization of ToxR (DiRita & Mekalanos, 1991). This mechanism of an extracellular protein causing a transmembrane protein to complex and activate transcription is fairly novel among bacterial transcriptional regulation systems.
  • Summary:
    • ToxR found in regulation of ctx operon (normally in Vibrio cholerae), which includes a gene for the toxin itself (ctx)
    • 3 regulator proteins are involved in the pathway:
      • ToxS: TM protein, only in periplasm
      • ToxR: TM protein, 1/3rd in periplasm, 2/3rds in cytoplasm
      • ToxT: inside cell (cytoplasm)
    • ToxS detects external environment (temp, pH, O2, osmolarity) via unknown mechanism. ToxR also does this in a limited way.
    • When activated, ToxS helps dimerize ToxR (mechanism does NOT include phosphorylation). ToxR's TM region winds up around each monomer (like a zipper)
    • When dimerized, ToxR's DNA binding domain causes transcription of ctx operon and also of ToxT gene
    • ToxT gene (along with ToxR) activates transcription of other proteins.
    • Structure of ToxR:
      • 294 aa (mw: 32,527 Da --> Miller et al. 1987)
      • Carboxy terminus: periplasm
      • Amino terminus: cytoplasm
      • TM region: Leu 183-Glu 198 (surrounded by Arg 182 on N-terminus and 3 uncharged aa on C terminus
      • Binding site known (Pfau & Taylor, 1996): TTTTGAT is tandemly repeated 8 times at -56 and once at -20 --> but these sequences are NOT enough! lacZ with these sequences will not produce B-gal.

• Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR.
  • 1984 paper, mostly stuff we know, but in being sensitive to manipulating cells that can have negative human affects: "Southern blot analysis suggests
  • that all V. chokrae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element." No, We Don't Have to Work With Virulent Cholera To Use ToxR.
• Invivo evidence for TonB dimerization with ToxR
  • I've only read the abstract, but TonB is a protein on the surface of E.Coli
  • Dimerization of ToxR causes ctx to be produced. But in E.Coli, this dimerization is studied by placing a lacZ (or other reporter gene) under the ctx promoter. So we don't need to be concerned about virulence factors.
• Co-ordinate expression of virulence genes by ToxR in Vibrio cholerae.
  • take home message: ToxR directly promotes some stuff (which includes production of ToxT). ToxT then directly promotes other stuff. ToxR can be a stand alone transcriptional factor.
• Changes in the periplasmic linker and in the expression level affect the activity of ToxR and lambda-ToxR fusion proteins in Escherichia coli.
  • Only read the abstract
  • These guys used ToxR-lambda phage repressor as a dimerization reporter
  • they constructed plasmids for various transmembrane fusion proteins.
  • Current status: reading it to summarize
  • Uh oh, problem: this article says: "the authors suggested that dimerization is not a requirement for ToxR activity in E. coli 10. Someone should get on the referenced paper right now and find out why this is a problem.
    • best I can tell, the reference claims that the ToxR periplasmic domain could be replaced with a few other dimers and a toxR-like monomer. Further analysis showed that this substitution only sort of worked. The "correct" ToxR conformation may be more important for function says the paper. This point is worth remembering, but likely not a game-breaker. It may be necassary to eliminate the possible substitutions in lab, but it would be better to know if this study manipulated the toxR itself significantly to use these substitutions. I'm inclined to think they did manipulate the protein significantly...

Chemotaxis

http://www.rowland.harvard.edu/labs/bacteria/images/fret1.jpg

• Information
  • MCPs:

MCPs are primary receptors for amino acids and secondary receptors for other attractants. They're not involved in transport and their genes are within the tar and tapE genes or in tsr and trg in region II (region II and III are coding regions contain the motility and chemotaxis genes).

There are 4 types of MCPs.

1)tsr has tsrE and tsrS, primary receptor and transducer for Serine, external pH, weak acid repellents, temperature, hydrophobic amino acids, and indole.

2)tar has tarE and tarS, primary receptor and transducer for aspartate, Co ion, Ni ion; secondary receptor and transducer for maltose.

3)trg has trgE and trgS, receptor and transducer for ribose and galactose

4)tap has tapE, receptor for dipeptides

5)tip has tipS, mediates responds

• • Sensory transduction components:

Communication between MCP receptors/transducers and the motor is done through CheA, CheB, CheR, CheW, CheY, and CheZ. All fall in region II.

Motility genes: there are 2 involved - motA and motB

Chemosensory system

Chemotactic signals detected by chemoreceptors (methyl-accepting chemotaxis proteins - MCPs)

Through CheW, adapter protein, MCPs link to CheA and phosphorylate it

CheA-P phosphorylate either CheY or CheB

CheY-P binds to Flim and causes turning of ecoli

If [attractant] decreases, MCPs phosphorylate CheA --> CheA-P phosphorylates CheY: as [CheY-P] increases see cell tumbling

CheY-P dephosphorylated by CheZ: responds to increasing [CheY-P]

When CheY-dephosphorylated, Che-B phosphorylated by CheA-P: CheB-P demethylates MCP and resets the sensor of attractant

If [attractant] increases, CheA phosphorylation inhibited and [CheY-P] decreases and motor switching decreases: smooth swimming

Papers on Chemotaxis/Dimerization:

Receptor signaling: Dimerization and beyond

[[../TarSummary1/]]

Role of alpha -Helical Coiled-coil Interactions in Receptor Dimerization, Signaling, and Adaptation during Bacterial Chemotaxis*

Activation of Bacterial Porin Gene Expression by a Chimeric Signal Transducer in Response to Aspartate

Dimerization is required for the activity of the protein histidine kinase CheA that mediates signal transduction in bacterial chemotaxis

MAKING SENSE OF IT ALL: BACTERIAL CHEMOTAXIS

Summary

CheA is the chemotaxis histidine protein kinase (HPK) however, it is cytoplasmic and senses through transmembrane receptors

The Chemoreceptors (MCPs)

Ecoli has 4 MCPs which form homodimers that are membrane spanning

Ligands bind to the periplasmic domain of MCPs between the 2 monomers of the dimer

Ligand binding alters the interactions between the periplasmic domains of the MCPs and change the interations in the MCP dimer

a 1.4Angstrom 'piston-like movement' of one of the helices

change of the periplasmic domains on ligand binding also alters the packing of the highly conserved cytoplasmic domains to cause signalling

signalling domain contains two adaptation regions

Tar recruits the methyltransferase CheR

it has been indicated in vivo that different receptors must be close enough to interact

Localization of methyl-accepting chemotaxis proteins

MCPs cluster in patches

MCPS might pack as trimers of dimers

Packing pattern would allow the necessary CheW and CheA interactions at the base of the MCPs

Receptors cluster are composed of signalling teams, a mixed trimers of MCP homodimers with different MCPs to regulate CheA

Max kinase activity seems to occur around 3.4 MCP to 1 CheW and 2 CheA

Variation in EColi

other MCP-like proteins exist: Aer--> senses for oxygen concentrations

Other Proteins in this pathway

CheW has 2 SH3 like subdomains and is both signallind for MCP and CheA

Forms the MCP-CheW-CheA complex

CheA works as a dimer

CheA shows increased autophosphorylation in response to a decrease in attractant binding or an increase in repellent binding

CheZ is a phosphatase and signal termination

Integrin Signalling

Picture Integrin signalling controls growth.

In Homo sapiens and mouse

TGF-Beta

Picture This pathway is in H. Sapiens.

Omp

e. coli

OmpR picture from 2004 UT project

Cytokine

Mammalian, S. Cerevisiae

Cytokines are small protein molecules that are the core of communication between immune system cells, and even between these cells and cells belonging to other tissue types. Cytokines act by binding to their cell-specific receptors. These receptors are located in the CELL MEMBRANE and each allows a distinct signal transduction cascade to start in the cell, that eventually will lead to biochemical and phenotypical changes in the target cell.

Most cytokine receptors lack intrinsic kinase activity. They are thought to transmit their regulatory signals primarily by the receptor-associated JAK (Janus kinase) family of tyrosine kinases. Ligand-binding to the receptor leads to JAK activation that phosphorylates cytoplasmic STAT (signal transducer and activator of transcription) proteins. Following phosphorylation on tyrosine residues, STATs are dimerized (resulting from phosphotyrosine - SH2 domain association). This dimerization is accompanied by translocation of STAT to the nucleus that results in DNA binding to specific response elements, and stimulation of gene transcription.

http://bio.winona.edu/bates/Immunology/images/12_07.gif	http://bbid.grc.nia.nih.gov/geneimages/2.Cytokine_Receptors.jpeg

Fec

• Surface signaling: novel transcription initiation mechanism starting from the cell surface
  • FecA is a SURFACE protein/receptor. Surface as in outermembrane. It moves Fe3+ into the cell and trinscribes itself. The protein domain directly involved in the induction half of the pathway has been identified. Does is only work in conjunction with the transport side? I don't know. It is a conformational change receptor - something that makes the kids uneasy. It may be possible to highjack the machinerey and engineer the binding site. It would be a serious hack job to make it work. Are you a pirate?

anyone into flies?

• bunch of Drosophilia pathways ~25 tyrosine kinase pathways, some are rather obscure (i.e. the pathways are not super-crucial to regular life).

no, noone is into flys