ras related c3 botulinum toxin substrate 1 rac1 n.
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Ras-related C3 botulinum toxin substrate 1 (RAC1)

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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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rac1 synopsis
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)

cell movement
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)

structure switch i ii regions
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)

structure insert region
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)

rac1 compared to rab5
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)

structure c terminus
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)

rac1 activation
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)

rac1 interaction tiam1 gef
RAC1 Interaction TIAM1 (GEF)

PDB ID: 1foe

Rac1 (red)

Switch Regions (green)

Tiam1 (blue)

DH Domain

(6)

cell adhesion and rac1
Cell Adhesion [and RAC1]

PLASMA MEMBRANE

E-cadherin

Tiam1

IQGAP1

RAC1

beta- catenin

Actin Filaments

beta- catenin

RAC1

alpha- catenin

beta- catenin

CYTOSOL

cell adhesion cont
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)

rac1 in nadph complex
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)

nadph complex
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)

superoxide significance
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)

rac1b
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)

rac1b structure comparison
RAC1B Structure Comparison

http://www.jbc.org/cgi/content/full/279/6/4743

Rac1b:GDP (purple) Rac1b:GppNHp (red) Rac1:GppNHp (brown)

(2)

rac1b properties
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)

rac1b as a biomarker
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)

treatment
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)

references
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>.