Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1.

102

Transforming growth factor-␤1 (TGF-␤1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-␤1 propeptide (latency-associated peptide (LAP)), and latent TGF-␤-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-␤1 complex. Transforming growth factor-␤1 (TGF-␤1) 2 is the prototypical member of a large family of structurally related proteins, with well documented roles in cellular proliferation, differentiation, apoptosis, adhesion, and extracellular matrix deposition (1, 2). Like other family members, TGF-␤1 is synthesized as a large precursor molecule consisting of an N-terminal prodomain (latency-associated peptide (LAP)) and a C-terminal mature domain. Within this precursor, LAP acts as a multifunctional peptide capable of regulating the crucial roles TGF-␤1 plays throughout development and in the maintenance of tissue homeostasis in adult life (3-5).Initially, hydrophobic residues near the N terminus of LAP bind to mature TGF-␤1 (6), and this interaction is necessary to maintain the molecule in a conformation competent for dimerization. Two precursors are then covalently linked at sites within both the mature growth factor and LAP to form the small latent complex (SLC) (7-10). The SLC can be cleaved by proprotein convertases (11, 12), but LAP remains non-covalently associated with the mature TGF-␤1 dimer (13,14). During the secretory process, LAP also interacts covalently with latent TGF-␤-binding proteins (LTBPs) to form the large latent complex (LLC), and it is in this form that TGF-␤1 is secreted from the cell (7, 15). In the absence of LTBPs, the reactive cysteine (Cys 33 ) within LAP forms an incorrectly paired disulfide bond with a free cysteine in mature TGF-␤1, ensuring the SLC is secreted in an inactive form (10).Extracellularly, LAP confers latency to TGF-␤1 by shielding the type II receptor-binding epitope on the outer convex surface of the mature dimer (16, 17). Lacking additional secreted antagonists, this is the major point of regulation of TGF-␤1 biological activity. Interestingly, of the 33 TGF-␤ ligands, only TGF-␤1, -␤2, -␤3, myostatin, and GDF-11 (growth and differentiation factor-11) bind their propeptides with high enough affinity to confer latency (16, 18 -21). For other family members (e.g. activins and bone morphogenetic proteins), affinity for receptors is greater than affinity for propeptides, and they are secreted in an "active" form (6,22).LTBPs associate with LAP via signature 8-Cys domains, which are unique to these proteins and the structurally related molecules, fibrillin-1 and fibrillin-2 (23-25). Once secreted, LTBPs target latent TGF-␤1 to fibrillin microfibrils within the extracellular matrix. TGF-␤1 signaling is dependant upon liberation of the mature ligand from the LLC, a process mediated by activators, such as thrombospondin-1 and integrins, that bind to specific residues in LAP ( 54 LSKL and 244 RGD, respectively) (4, 26 -28). By altering the co...

“…ings (4,6,14) that demonstrated that the N terminus of LAP interacted with the TGF-␤1 dimer to mediate growth factor assembly, secretion, and latency.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Two Distinct Regions of Latency-associated Peptide Coordinate Stability of the Latent Transforming Growth Factor-β1 Complex

Walton¹,

Makanji²,

Chen

et al. 2010

102

“…It is now clear that LAP is implicated at different levels in TGF␤ biological activity. Previous studies have indicated that LAP plays important roles in secretion and proper folding of the mature growth factor molecule (5,11,12). Furthermore, specific binding of LAP to TGF␤ may be an important mechanism in regulating the biological functions of secreted TGF␤ (6,13).…”

mentioning

confidence: 99%

Analysis of Small Latent Transforming Growth Factor-β Complex Formation and Dissociation by Surface Plasmon Resonance

Bailly

Brand

Chambaz

et al. 1997

Transforming growth factor-␤ (TGF␤) is a pluripotent regulator of cell growth and differentiation. The growth factor is expressed as a latent complex that must be converted to an active form before interacting with its ubiquitous high affinity receptors. This conversion involves the release of the mature TGF␤ through disruption of the noncovalent interactions with its propeptide or latency associated protein (LAP). Complex formation or dissociation between LAP and TGF␤ plays a very important role in TGF␤ biological activity at different steps. To further characterize the kinetic parameters of this interaction, we have employed surface plasmon resonance biosensor methodology. Using this technique, we observed real time association of LAP with mature TGF␤1. The complex formation showed an equilibrium K d around 3-7 nM. Furthermore, we observed dissociation of the complex in the presence of extreme pH, chaotropic agents, or plasmin, confirming their effects on TGF␤ activation. The same approach was used to examine whether latent TGF␤1 could interact with thrombospondins, previously described as activators of latent TGF␤. Using this method, we could not detect any direct interaction of thrombospondins with either LAP alone, TGF␤1 alone, or the small latent TGF␤1 complex. This suggests that activation of latent TGF␤1 complex by thrombospondins is through an indirect mechanism.

“…Sha et al (17) used deletion and insertion mutagenesis to identify regions of the propeptide important for secretion of biologically active mature TGF-␤1 and determined that amino acids between residues 50 and 80 interact with the mature peptide in the latent complex form of TGF-␤1. Elimination of all glycosylation sites on the TGF-␤1 and -␤2 propeptide prevents secretion of any mature protein (18,19), and mutation of the TGF-␤1 propeptide cysteine residues results in the secretion of differently assembled and processed forms (20).…”

Section: Macrophage Inhibitory Cytokine-1 (Mic-1)mentioning

confidence: 99%

The Propeptide of the Transforming Growth Factor-β Superfamily Member, Macrophage Inhibitory Cytokine-1 (MIC-1), Is a Multifunctional Domain That Can Facilitate Protein Folding and Secretion

Fairlie

Zhang

et al. 2001

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-␤ (TGF-␤) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-␤1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major ␣-helix in the mature protein, residues 56 -68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-␤1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-␤ superfamily protein folding.