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Ras-related C3 botulinum toxin substrate 1 (RAC1)

Ras-related C3 botulinum toxin substrate 1 (RAC1). Aksam Ahmad. RAC1 Synopsis. small G protein (GTPase) member of the Ras superfamily of guanosine triphosphate (GTP) binding proteins cell functions: regulation actin polymerization in membrane ruffling

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Ras-related C3 botulinum toxin substrate 1 (RAC1)

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  1. Ras-related C3 botulinum toxin substrate 1 (RAC1) Aksam Ahmad

  2. RAC1 Synopsis • small G protein (GTPase) • member of the Ras superfamily of guanosine triphosphate (GTP) binding proteins • cell functions: • regulation actin polymerization in membrane ruffling • formation of focal adhesion (FA) complexes • formation of lamellipodia • regulation of NADPH oxidase activity in NADPH complex (4)

  3. CELL MOVEMENT • essential element of many cellular processes such as phagocytosis, wound repair, embryogenesis, immunological responses to tumor metastasis • critical step is extension of protrusions in the direction of the desired move, which is accomplished by membrane ruffling and the formation of lamellipodia • Rac1 is the primary Rho-GTPase responsible for regulating the cytoskeletal remodeling involved with lamellipodia dynamics (1)

  4. Structure: Switch I/II Regions • responsible for molecular interactions • Switch I mainly interacts with downstream effectors, such as IQGAP1 • also called ‘effector region’ • Switch II mainly interacts with activating proteins (GEFs) Switch II Switch I PDB ID: 1mh1 (4)

  5. Structure: Insert Region • only present in Rho family (residues 124-135) • between beta-strand 5 and alpha-helix 4 • essential for mitogenesis and apoptosis • regulating downstream effector interactions, specifically NADPH oxidase insert region PDB: 1mh1 (3)

  6. RAC1 compared to RAB5 • Rab5 is also a member of the Ras superfamily of GTP binding proteins • Whereas Rac1 belongs to the Rho subfamily, Rab5 belongs to the Rab subfamily • Rab subfamily is largest subfamily of Ras GTP proteins • All Ras proteins have nearly identical structure, except for Rho subfamily • INSERT REGION only present in Rho family insert region Rac1: 1mh1 (red) Rab5: 1r2q (purple) (3)

  7. Structure: C-Terminus • C-terminus participates in the binding of Rac1 to the membrane • necessary particularly in NADPH complex • members of Rho subfamily (and to lesser extent Ras superfamily) share ~92% sequence homology; sequence divergence occurs mainly in C-Termini • Rac1 contains polybasic amino acid residues while other Rho proteins are less basic (4)

  8. RAC1 Activation • Rac1 activation is regulated by guanosine nucleotide exchange factors (GEFs) • Tiam1 is one such GEF • Dependent on replacing bound GDP with GTP, as Rac1 is initially bound to GDP in its inactive state • GTP-bound Rac1 (active) is able to interact with downstream effectors • GTP-ase activating proteins (GAPs) facilitate GTP hydrolysis necessary for switch from inactive to active Rac1 • RhoGDI regulates inactive/active cycles by isolating GDP-Rac1 RAC1 RAC1 GDP GTP active inactive (6)

  9. RAC1 Interaction TIAM1 (GEF) PDB ID: 1foe Rac1 (red) Switch Regions (green) Tiam1 (blue) DH Domain (6)

  10. Cell Adhesion [and RAC1] PLASMA MEMBRANE E-cadherin Tiam1 IQGAP1 RAC1 beta- catenin Actin Filaments beta- catenin RAC1 alpha- catenin beta- catenin CYTOSOL

  11. CELL ADHESION [cont.] ACTIN FILAMENTS linked to membrane through VINCULIN VINCULIN links to E-CADHERIN with the ALPHA/BETA-CATENINS E-CADHERIN essentially plays biggest role in ‘gluing’ cells together http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/actin.htm (1)

  12. RAC1 in NADPH Complex • Consists of p40phox, p47phox, p67phox, b558 (membrane-bound heterodimer composed of gp91phox and p22phox) and Rac1 • Activation of complex occurs when cell is exposed to inflammatory stimuli and phagocytes are activated • p47phox & p67phox are phosphorylated (SO4); Rac1 is activated (becomes GTP bound) • These three components then translocate to membrane and form complex with b558 • Complex then initiates electron flow and superoxide production (5)

  13. NADPH Complex • Note: • p67phox and p47phox are phosphorylated • Rac1 is in its active form bound to GTP • Rac1 is bound to membrane via C-Terminus • b558 is composed of p22phox and gp91phox • SIGNIFICANT RESULT: • superoxide is generated p47phox p67phox b558 membrane Rac1 p40phox SO4 GTP NADPH + 2O2 ↔ NADP+ + 2O2°- + H+ (5)

  14. Superoxide Significance • facilitates destruction of invading microorganisms during phagocytosis • capable of killing bacteria and fungi due to its ability to react with other compounds • Flipside: • can lead to increased production of reactive oxygen species (ex: hydrogen peroxide), which cause protein oxidation, DNA damage, and neuronal cell death (5)

  15. RAC1B • novel splice variant of Rac1 • different from mutation; alternative sequences that can occur naturally • contains extra 19 amino acid insertion (between codons 75 and 76 and end of Switch II region) • associated with tumors and human cancers.. Switch I Regions Rac1: 1mh1 (purple) Rac1b: 1ryf (yellow) (2)

  16. RAC1B Structure Comparison http://www.jbc.org/cgi/content/full/279/6/4743 Rac1b:GDP (purple) Rac1b:GppNHp (red) Rac1:GppNHp (brown) (2)

  17. RAC1B Properties • expressed in human breast and colon carcinoma cells • impaired GTPase activity and interaction with downstream effectors; no interaction with Rho GDI • causes increase in cellular reactive oxygen species (ROS)  stimulate expression of transcription factor Snail and EMT  cause oxidative damage to DNA and genomic instability leading to cancer • promotes growth of NIH3T3 cells (cells that maintain growth of extracellular matrix of tissues) • not involved in all important lamellipodia formation, which therefore impairs cell mobility (2)

  18. RAC1B as a Biomarker • Rac1b has been identified as a promising early stage biomarker for matrix metalloproteinase (MMP)-induced malignancies • such malignancies include breast, colon, prostate, kidney, skin, stomach, ovary, and lung cancers • biomarker is any substance that serves as an indicator of biologic state • Rac1b is NOT generated in non-malignant cells • unlike most uncogenic isforms induced in cancers, Rac1b becomes more highly activated (2)

  19. Treatment • Rac1b associated malignancies are highly therapeutic • Labs (including Bissell Lab at Berkeley) have been able to develop Rac1b antibody • Rac1b siRNA developed as well • involved in the RNA interference (RNAi) pathway and more specifically interferes with expression of specific gene (2)

  20. REFERENCES • (1) "Actin Cytoskeleton". April 20, 2008 <http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/ part1/actin.htm>. • (2) Fiegen, Dennis. "Alternative Splicing of Rac1 Generates Rac1b, a Self-activating GTPase". April 20, 2008 <http://www.jbc.org/cgi/content/full/279/6/4743>. • (3) Freeman, Jennifer. "Rac Insert Region Is a Novel Effector Region That Is Implicated in the Activation of NADPH Oxidase". April 20, 2008 <http://www.jbc.org/cgi/content/abstract/271/33/19794>. • (4) "RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1". April 20, 2008 <http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602048>. • (5) Williams, Emily. "Human Rac1 in Complex with p67phox". April 20, 2008 <http://biology.kenyon.edu/BMB/Chime2/2003/RAC1/FRAMES/index.htm>. • (6) Worthylake, David. "Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1". April 20, 2008 <http://www.nature.com/nature/journal/v408/n6813/full/408682a0.html>.

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