Pyrowolakis TGFbeta/ BMP signaling

Heterodimerization of Type I and Type II receptor upon ligand binding -> crossphosphorylation (type II phosphorylates type I at G/S domain) and recruitment and Phosphorylation of R-SMAD -> recruitment of Co-SMAD enabling nuclear localisation -> binding to SMAD response elements (SBEs binding motives on DNA) -> target gene expression in various forms

• SMAD response elements -> DNA binding motives

• gene expression modes:

  • self-enablling (expression of protein that acts as activator or repressor Co-Factor for other genes

  • Switch enhancer: activation or repression depending on chromatin state, Co-factor availability -> Cell type dependant!!!

  • Derepression: repressor binding Co-Factor is degraded upon TGF-ß signaling -> gene activation

interference on the level of:

• Ligands - regulation bioavailability of ligand -ligand traps (LAP), acessory receptors

• Receptors - Degradation, Blocking Phosphorylation, Pseudoreceptors

• R-Smads

• Co-Smads, Co-Factors (e.g. DNA binding)

• Transport, Gene transcription

e.g. iSmads -> block access of R-Smad to Receptors • channel activated Receptors to degradation • prevent R-Smads binding to co-Smad • prevent Smad function in the nucleus

Other than that interferance can occur through cross talk with other signaling pathways e.g. FGF/RAF/RAS/MEK/ERK inhibiting SMADs or WNT / GSK3

• encoding genes identified accross species such as fruit flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans), and various vertebrates (mice, fish, and birds)

• AA Sequence homology

• Similar structure (folding, crystal structure, functions, mechanism)

• Functional conservation -> similar roles and even cross species functional studies (use the components from one organism in another —> Dpp from Drosophila in humans can substitue BMP4

• Same mechanism of the signal transduction

• Phylogenetics (Blast & sequence comparison)

• phenotype rescue experiments in eg drosophila or c elegans with human tgf or bmp

Target the receptor type I GS domain -> deletion, knock down

Target SMADs by mutations in their phosphorylation site (iSMADS)

many other methods

transgenes: conditional allele with flanked stop cassette in front of TGFb/ iSmad coding region

Activate:

- bead with ligand, conditional knockout, dimerization receptor w/o ligand e.g by ab, phosphomimetic receptors mimicks conformational change of ligand binding

Inhibit:

- delete GS domain, substitution with mutant receptor (dominant negative), ligand traps, iSmads

BMP ligands act as morphogens -> secreted from a source form a concentration gradient depending on a threshhold different cell fates are determinded

Uniform ligand expression -> graded signaling by e.g Sog

also possible by inhibitors or ligand traps (reverse gradient)

Gradient formation by:

diffusion, serial transfer, lipoprotein particels or direct transfer

That its consistant with reduction of BMP signal is easily explained since this is their function as BMP inhibitors but that its a similar phenotype to hyperactivation is a little more complicated and has to do with the BMP gradient threshold, since in the Chordin/Sog mutant the BMP ligands can diffuse everywhere without inhibition the concentration cant spike in the specific region meaning the threshold to induce cell differentiation is not reached -> same effect as if the BMP signal was reduced.

• hyperactivation: chordin/sog shape the BMP gradient. if shaping activity is lost, BMP diffuses more ventrally and cause hyperactivation in those regions where it usually does not signal

• reduction: General amount of BMP is not affected. since it can diffuse further, the concentration thresholds in regions where it is usually active cannot be reached

Mangold used newt(salamander) embryos (Triton taeniatus and Triton cristatus) for her experiments. These species were chosen because they have easily distinguishable pigmentation, which allowed for clear identification of transplanted tissues -> distinguish cells, visualizing the results

For the experiment in the newt gastrula stage the dorsal lip tissue of the blastopore was transplated to the ventral side of a host embry and two body axis developed

transplanted tissue able to induce surrounding unpigmented host tissue to form second embryo -> organizing capability -> Spemann Organizer

Gene Knockouts/Knockdowns: Use CRISPR/Cas9 or RNA interference to knock out or knock down genes encoding potential intermediate signaling molecules. If the BMP effects are direct, knocking out these intermediates should not abolish the BMP-induced responses.

Conditional Mutants: Employ tissue-specific or inducible gene knockout models to remove BMP receptors or SMAD proteins in target tissues. Direct effects should be absent in tissues lacking these components.

Ligand Binding Studies: Use labeled BMP ligands (radioactive or fluorescent) to track their binding to receptors on cells located at various distances from the BMP source. Direct effects would be indicated by the presence of labeled ligand-receptor complexes on distant cells

Receptor Blockade: Apply specific BMP receptor antagonists or neutralizing antibodies at different locations. If BMP effects are direct, blocking the receptors should inhibit the response.

-> Same with a physical barrier

Tissue Grafting: Transplant tissue sources of BMP ligands at various positions in an embryo or tissue culture. Observe the effects on distant target tissues. If the effects are direct, transplanted tissues should induce responses without the need for intermediate signals. -> Mangold

• three possible mechanisms: direct mechanism, signal relay and epigenetic inheritance

• direct vs. signal relay:

  Flp transgene: frt-flanked GFP as stop cassette in front of constitutively active dpp receptor. Area of omb (target gene of dpp) exactly overlaps with ectopic expression of constitutively expression of dpp receptor which means ist must act in a direct mechanism. If it would act in a signal relay mechanism, the area of omb expression would be larger than the area of the constitutively active receptor. Signal relay means that dpp signaling causes dpp expression?

• direct vs. epigenetic inheritance:

  usage of mitotic clones. GFP gets activated in all cells that respond to dpp, marker gets inherited by progenies. (???)

  usage of loss of function receptor. Expression of homozygous loss of function receptor only in progenies. If dpp would act via cellular memory response omb would still be expressed in cells with loss of function receptor, but it is not. This means that constant dpp is needed —> direct mechanism!

Formation of receptor complexes with other receptor (tyrosine kinase from FGFR)

SMAD interactions with other TF -> repressor / activator function

Posttranslational modification of Smads can alter TGFß/BMP signaling -> phosphorylation, Ubiquitination

Feedback regulators

Epigenetic modifieres

• Regulation common targets

• regulation availability of BMP/TGFß regulators

• regulation core components of the pathway

• E.g WNT, FGF at level of Smad activity -> Smads act as platforms for different kinases & mediates different effects

In a normal environment suppressor activity dominates but given the right circumstances, transcription factors, loss of adherence [EMT] -> the BMP signaling leads to pro-oncogenic activity

Important is the change in genetic / epigenetic context

Target the ligand or block the receptors -> with small molecule inhibitors or antibodies

To only target the aberrant signaling more downstream target is maybe better but needs very much information / reseach-> target the Co-Factors that are responsible for the aberrant signal

Target the cross talk with other pathways or combine therapies

Target tissue specific BMP signaling with gene editing or targeted delivery with nanoparticals or liposomes

small molecule inhibitor of Co-Smad or R-Smads?