TGFβ ACTIVATION AND SIGNALING

Presented by: Fathimath Shibana M. Sc Biotechnology 3rd Semester, 2013 University of Mysore

Guided by: Dr. Geetha N.P . Assistant Professor DOS in Biotechnology

WHAT IS TGF?• It is transforming growth factor or tumor growth factor.• There are two types, TGF-α and TGF-β• TGF-α is a single growth factor which is upregulated in some

human cancers. It is produced in macrophages, brain cells and keratinocytes (basal cells in epidermis) and induces epithelial development.

• TGF-β is a diverse family of growth factor. Many different members and subfamilies are within it. TGF-β is a protein that controls proliferation, cellular differentiation, and other functions in most cells.

• It is a type of cytokine which plays a role in immunity, cancer, bronchial asthma, heart disease, diabetes, Marfan syndrome, etc

• In human TGF-β exist in 3 subtypes. They are TGF-β1, TGF-β2 and TGF-β3 which upregulates Marfan syndrome

STRUCTURE OF TGFβ

Crystal Structure of TGF-beta 1 Crystal Structure of TGF-beta 2 Crystal Structure of TGF-beta 3

Structure of TGF-β consists of 3 domains.1. N-terminal domain which associate with the precursor molecule

to proper cellular secretory pathways2. Peptide domain which support folding or dimerization of

mature cytokine3. C-terminal domain which are approximately 100-114 amino acid

long, they help autocrine signaling molecule which is highly conserved across the superfamily.

TGFβ ACTIVATION• Some currently known factors which activates TGF-β are

Protease, Integrins, PH and oxygen reactive species (ROS)

• Disruption of these activating factors can leads to unregulated TGF-β signaling levels that may cause several complications including inflammation, autoimmune disorders, fibrosis, cancer and cataracts.

• In most cases activated TGF-β ligand will initiate TGF-β signaling cascade as long as TGF receptors I and II are within reach. This is due to high affinity between TGF-β and its receptors.

TGFβ Activation continued.....

LATENT TGFβ COMPLEX• TGF-β1, TGF-β2 and TGF-β3 are synthesized as Precursor

molecule which contains propeptide region in addition to TGF-β homodimer.

• After synthesized TGF-β homodimer interact with Latency Associated Peptide (LAP). [LAP is a protein derived from N-terminal region of TGF-β gene product].

• Interaction of TGF-β homodimer with LAP forms Small Latent Complex (SLC)

• This complex remains in cell unit is bounded by another protein called Latent TGF-β Binding Protein (LTBP) forming large complex called Large Latent Complex (LLC)

• It is LLC that get secreted to Extracellular Matrix (ECM)

TGFβ Activation continued.....Latent TGFβ Complex continued.....

• In most cases before LLC is secreted, TGF-β precursor is cleaved from propeptide but remains attached to it by non-covalent bonds .

• After its secretion it remains in ECM as inactive complex containing both LTBP and LAP which needs to be further processed to release active TGF-β.

• The attachment of TGF-β to LTBT is by disulphide bond which allow it to remain inactive by preventing it from binding to its receptors.

• Because of different cellular mechanism require distinct levels of TGF-β signaling, the inactive complex of this cytokine gives opportunity for proper mediation of TGF-β signaling.

INTEGRIN DEPENDENT TGFβ ACTIVATIONTGFβ Activation continued.....

1. ACTIVATION BY PROTEASE AND METALLOPROTEASE

• Plasmin and no. of Matrix Metalloprotease (MMP) play a key role in promoting tumor invasion and tissue remodeling by inducing proteolysis of several ECM components.

• TGF-β activation process involves release of LLC from matrix, followed by proteolysis of LAP to release TGF-β to its receptors

• MMP9 and MMP2 are known to cleave TGF-β• LAP complex contains protease sensitive hinge region which can

be target for liberation of TGF-β. Despite the fact that MMP plays a key role in activating TGF-β, mice with mutation in MMP9 and MMP2 gene can still activate TGF-β and do not show any TGF-β deficiency phenotypes. It may be due to unknown proteases.

TGFβ Activation continued.....Integrin dependentTGFβ activation continued.....

2. ACTIVATION BY PH• Acidic condition can denature the LAP.• Treatment of medium with extremes of pH (1.5 or 12) result in

activation of TGF-β as shown by radio receptor assays.• Mild acidic treatment (pH 4.5) yielded only 20-30% of the

competition achieved by pH 1.5

3. ACTIVATION BY REACTIVE OXYGEN SPECIES (ROS)• LAP structure is important to maintain its function• Structure modification of LAP leads to disturbing the interaction

between LAP and TGF-β and thus activates it.• Factors that may cause this modification may include hydroxyl

radicals from ROS. TGF-β was rapidly activated after in vivo radiation exposure ROS

Fig: The construction and extracellular activation of latent TGF-β1. The TGF-β1 dimer is synthesized intracellularly and combines with LAP to form a latent

precursor (SLC). The SLC then forms a LLC with LTBP-1 via covalent bonds. The hollow arrows show the activating sites of TGF-β1. Proteases and integrins can

activate latent TGF-β1 via direct proteolytic cleavage and/or by inducing a conformational change in LAP.

TGFβ SIGNALING PATHWAYMECHANISM

Ligand binding Receptor recruitment and phosphorylation SMAD phosphorylation

Co-SMAD binding Transcription

TGFβ SIGNALING PATHWAY

• TGF-β signaling pathway begins with a TGF-β dimer binding to Type II TGF-β receptor at cell surface.

• TGF-β dimer induce formation of complex between type II and type I TGF-β receptors, both of which are transmembrane serine/threonine kinases.

• Once TGF-β dimer binds to type II receptor, type II receptor phosphorelates and activates type I receptor.

• The activated type I receptor phosphorelates a receptor regulated Smad (R-smad) which then dimerizes with a Co-smad.

• The smad dimer translocates into the nucleus and with a DNA binding partner activates transcription of target genes.

BIBLIOGRAPHY• http://en.wikipedia.org• http://www.sinobiological.com/TGF-beta-Structure-and-

Function-a-5894.html • http://maptest.rutgers.edu/drupal/?q=node/131 • http://www.karger.com/Article/FullText/338799 • http://jkweb.berkeley.edu/external/pdb/2001/tgf_beta_R1/

signaling_schematic.jpg• http://bcs.whfreeman.com/lodish5e/content/cat_010/14010-

01.htm?v=category&i=14010.01&s=00010&n=14000&o=00PRS