Smads (or SMADs) comprise a family of structurally similar
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s that are the main signal transducers for receptors of the
transforming growth factor beta
Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-β 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other sign ...
(TGF-B) superfamily, which are critically important for regulating cell development and growth. The abbreviation refers to the homologies to the ''
Caenorhabditis elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (ro ...
'' SMA ("small" worm phenotype) and
MAD family ("Mothers Against Decapentaplegic") of genes in
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
.
There are three distinct sub-types of Smads: receptor-regulated Smads (
R-Smads), common partner Smads (Co-Smads), and inhibitory Smads (
I-Smads). The eight members of the Smad family are divided among these three groups.
Trimers of two receptor-regulated SMADs and one co-SMAD act as
transcription factors
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The func ...
that regulate the expression of certain genes.
Sub-types
The R-Smads consist of
Smad1
Mothers against decapentaplegic homolog 1 also known as SMAD family member 1 or SMAD1 is a protein that in humans is encoded by the ''SMAD1'' gene.
Nomenclature
SMAD1 belongs to the SMAD, a family of proteins similar to the gene products of the ...
,
Smad2
Mothers against decapentaplegic homolog 2 also known as SMAD family member 2 or SMAD2 is a protein that in humans is encoded by the ''SMAD2'' gene. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the ''Dros ...
,
Smad3
Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene.
SMAD3 is a member of the SMAD family of proteins. It acts as a mediator of the signals initiated by t ...
,
Smad5 and
Smad8/9,
and are involved in direct signaling from the TGF-B receptor.
Smad4
SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a highly conserved protein present in all metazoans. It belongs to the SMAD family of transcription factor proteins, ...
is the only known human Co-Smad, and has the role of partnering with R-Smads to recruit co-regulators to the complex.
Finally,
Smad6
SMAD family member 6, also known as SMAD6, is a protein that in humans is encoded by the ''SMAD6'' gene.
SMAD6 is a protein that, as its name describes, is a homolog of the Drosophila gene " mothers against decapentaplegic". It belongs to the SM ...
and
Smad7
Mothers against decapentaplegic homolog 7 or SMAD7 is a protein that in humans is encoded by the ''SMAD7'' gene.
SMAD7 is a protein that, as its name describes, is a homolog of the Drosophila gene: "Mothers against decapentaplegic". It belongs to ...
are I-Smads that work to suppress the activity of R-Smads.
While Smad7 is a general TGF-B signal inhibitor, Smad6 associates more specifically with BMP signaling. R/Co-Smads are primarily located in the cytoplasm, but accumulate in the nucleus following TGF-β signaling, where they can bind to DNA and regulate transcription. However, I-Smads are predominantly found in the nucleus, where they can act as direct transcriptional regulators.
Discovery and nomenclature
Before Smads were discovered, it was unclear what downstream effectors were responsible for transducing TGF-B signals. Smads were first discovered in ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
'', in which they are known as
mothers against dpp
Mothers against decapentaplegic is a protein from the SMAD family that was discovered in ''Drosophila''. During ''Drosophila'' research, it was found that a mutation in the gene in the mother repressed the gene decapentaplegic in the embryo. The ...
(Mad), through a genetic screen for dominant enhancers of
decapentaplegic
Decapentaplegic (Dpp) is a key morphogen involved in the development of the fruit fly ''Drosophila melanogaster'' and is the first validated secreted morphogen. It is known to be necessary for the correct patterning and development of the early ' ...
(dpp), the ''Drosophila'' version of TGF-B.
Studies found that Mad null mutants showed similar phenotypes to dpp mutants, suggesting that Mad played an important role in some aspect of the dpp signaling pathway.
A similar screen done in the ''
Caenorhabditis elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (ro ...
'' protein SMA (from gene ''sma'' for small body size) revealed three genes, Sma-2, Sma-3, and Sma-4, that had similar mutant phenotypes to those of the TGF-B like receptor
Daf-4
The DAF-4 gene encodes for the only type II receptor of TGF-beta signaling pathway in the worm ''Caenorhabditis elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil ...
. The human homologue of Mad and Sma was named Smad1, a portmanteau of the previously discovered genes. When injected into ''
Xenopus
''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-known ...
'' embryo animal caps, Smad1 was found to be able to reproduce the mesoderm ventralizing effects that
BMP4
Bone morphogenetic protein 4 is a protein that in humans is encoded by ''BMP4'' gene. BMP4 is found on chromosome 14q22-q23.
BMP4 is a member of the bone morphogenetic protein family which is part of the transforming growth factor-beta superfamil ...
, a member of the TGF-B family, has on embryos. Furthermore, it was demonstrated that Smad1 had transactivational ability localized at the carboxy terminus, which can be enhanced by adding BMP4. This evidence suggests that Smad1 is responsible in part for transducing TGF-B signals.
Protein
Smads are roughly between 400 and 500 amino acids long, and consist of two globular regions at the amino and carboxy termini, connected by a linker region. These globular regions are highly conserved in R-Smads and Co-Smads, and are called Mad homology 1 (MH1) at the N-terminus, and MH2 at the C-terminus. The MH2 domain is also conserved in I-Smads. The MH1 domain is primarily involved in DNA binding, while the MH2 is responsible for the interaction with other Smads and also for the recognition of transcriptional co-activators and co-repressors.
R-Smads and Smad4 interact with several DNA motifs though the MH1 domain. These motifs include the CAGAC and its CAGCC variant, as well as the 5-bp consensus sequence GGC(GC), (CG). Receptor-phosphorylated R-Smads can form homotrimers, as well as heterotrimers with Smad4 in vitro, via interactions between the MH2 domains. Trimers of one Smad4 molecule and two receptor-
phosphorylated
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, whi ...
R-Smad molecules are thought to be the predominant effectors of TGF-β transcriptional regulation.
The linker region between MH1 and MH2 is not just a connector, but also plays a role in protein function and regulation. Specifically, R-Smads are phosphorylated in the nucleus at the linker domain by CDK8 and 9, and these phosphorylations modulate the interaction of Smad proteins with transcriptional activators and repressors. Furthermore, after this phosphorylation step, the linker undergoes a second round of phosphorylations by GSK3, labelling Smads for their recognition by
ubiquitin ligase
A ubiquitin ligase (also called an E3 ubiquitin ligase) is a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin ...
s, and targeting them for
proteasome
Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases.
Proteasomes are part of a major mechanism by w ...
-mediated degradation. The transcription activators and the ubiquitin ligases both contain pairs of
WW domain
The WW domain, (also known as the rsp5-domain or WWP repeating motif) is a modular protein domain that mediates specific interactions with protein ligands. This domain is found in a number of unrelated signaling and structural proteins and may be ...
s. These domains interact with the PY motif present in the R-Smad linker, as well as with the phosphorylated residues located in the proximity of the motif. Indeed, the different phosphorylation patterns generated by CDK8/9 and GSK3 define the specific interactions with either transcription activators or with ubiquitin ligases. Remarkably, the linker region has the highest concentration of amino acid differences among metazoans, although the phosphorylation sites and the PY motif are highly conserved.
Sequence conservation
The components of the TGF-beta pathway and in particular, the R-Smads, Co-Smad and I-Smads, are represented in the genome of all metazoans sequenced to date. The level of sequence conservation of the Co-Smad and of R-Smads proteins across species is extremely high. This level of conservation of components -and sequences- suggests that the general functions of the TGF-beta pathway have remained generally intact ever since. I-Smads have conserved MH2 domains, but divergent MH1 domains as compared to R-Smads and Co-Smads.
Role in TGF-β signalling pathway
R/Co-Smads
TGF-B ligands bind receptors consisting of type 1 and type 2
serine/threonine kinase
A serine/threonine protein kinase () is a kinase enzyme, in particular a protein kinase, that phosphorylates the OH group of the amino-acid residues serine or threonine, which have similar side chains. At least 350 of the 500+ human prote ...
s, which serve to propagate the signal intracellularly. Ligand binding stabilizes a receptor complex consisting of two type 1 receptors, and two type 2 receptors.
Type 2 receptors then can phosphorylate type 1 receptors at locations on the GS domain, located N-terminally to the type 1 kinase domain.
This phosphorylation event activates the type 1 receptors, making them capable of further propagating the TGF-B signal via Smads. Type 1 receptors phosphorylate R-Smads at two C-terminal serines, which are arranged in an SSXS motif. Smads are localized at the cell surface by Smad anchor for receptor activation (SARA) proteins, placing them in proximity of type 1 receptor kinases to facilitate phosphorylation. Phosphorylation of the R-Smad causes it to dissociate from SARA, exposing a nuclear import sequence, as well as promoting its association with a Co-Smad. This Smad complex is then localized to the nucleus, where it is able to bind their target genes, with the help of other associated proteins.
I-Smads
I-Smads disrupt TGF-B signaling through a variety of mechanisms, including preventing association of R-Smads with type 1 receptors and Co-Smads, down-regulating type 1 receptors, and making transcriptional changes in the nucleus. The conserved MH2 domain of I-Smads is capable of binding to type 1 receptors, thus making it a competitive inhibitor of R-Smad binding. Following R-Smad activation, it forms a
heteromeric A heteromer is something that consists of different parts; the antonym of homomeric. Examples are:
Biology
* Spinal neurons that pass over to the opposite side of the spinal cord.
* A protein complex that contains two or more different polypeptide ...
complex with an I-Smad, which prevents its association with a Co-Smad. In addition, the I-Smad recruits a ubiquitin ligase to target the activate R-Smad for degradation, effectively silencing the TGF-β signal.
I-Smads in the nucleus also compete with R/Co-Smad complexes for association with DNA binding elements. Reporter assays show that fusing I-Smads to the DNA-binding region of reporter genes decreases their expression, suggesting that I-Smads function as transcriptional repressors.
Role in cell cycle control
In adult cells, TGF-β inhibits cell cycle progression, stopping cells from making the G1/S phase transition. This phenomenon is present in the epithelial cells of many organs, and is regulated in part by the Smad signaling pathway. The precise mechanism of control differs slightly between cell types.
One mechanism by which Smads facilitate TGF-B induced
cytostasis
Cytostasis (cyto – cell; stasis – stoppage) is the inhibition of cell growth and multiplication. Cytostatic refers to a cellular component or medicine that inhibits cell division.
Cytostasis is an important prerequisite for structured multic ...
is by downregulating
Myc
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' (MYC), ''l-myc'' ( MYCL), and ''n-myc'' (MYCN). ''c-myc'' (also sometimes refe ...
, which is a transcription factor that promotes cell growth. Myc also represses p15(Ink4b) and p21(Cip1), which are inhibitors of Cdk4 and Cdk2 respectively. When there is no TGF-β present, a repressor complex composed of Smad3, and the transcription factors E2F4 and p107 exist in the cytoplasm. However, when TGF-B signal is present, this complex localizes to the nucleus, where it associates with Smad4 and binds to the TGF-B inhibitory element (TIE) of the Myc promoter to repress its transcription.
In addition to Myc, Smads are also involved in the downregulation of Inhibitor of DNA Binding (ID) proteins. IDs are transcription factors that regulate genes involved in cell differentiation, maintaining multi-potency in stem cells, and promoting continuous cell cycling.
Therefore, downregulating ID proteins is a pathway by which TGF-B signaling could arrest the cell cycle. In a DNA microarray screen, Id2 and Id3 were found to be repressed by TGF-B, but induced by BMP signaling. Knocking out Id2 and Id3 genes in epithelial cells enhances cell cycle inhibition by TGF-B, showing that they are important in mediating this cytostatic effect. Smads are both a direct and indirect inhibitor of Id expression. TGF-B signal triggers Smad3 phosphorylation, which in turn activates ATF3, a transcription factor that is induced during cellular stress. Smad3 and ATF3 then coordinate to repress Id1 transcription, resulting in its downregulation. Indirectly, Id downregulation is a secondary effect of Myc repression by Smad3. Since Myc is an inducer of Id2, downregulating Myc will also result in reduced Id2 signaling, which contributes to cell cycle arrest.
Studies show that Smad3, but not Smad2, is an essential effector for the cytostatic effects of TGF-B. Depleting endogeneous Smad3 via RNA interference was sufficient to interfere with TGF-B cytostasis. However, depleting Smad2 in a similar manner enhanced, rather than halted, TGF-B induced cell cycle arrest. This suggests while Smad3 is necessary for TGF-B cytostatic effect, the ratio of Smad3 to Smad2 modulates the intensity of the response. However, overexpressing Smad2 to change this ratio had no effect on the cytostatic response. Therefore, further experiments are necessary to definitely prove that the ratio of Smad3 to Smad2 regulates intensity of cytostatic effect in response to TGF-B.
Smad proteins have also been found to be direct transcriptional regulators of Cdk4. Reporter assays in which
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'', ...
was placed under a Cdk4 promoter showed increased luciferase expression when Smad4 was targeted with
siRNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating wi ...
s. Repression of Smad2 and 3 did not have any significant effect, suggesting that Cdk4 is directly regulated by Smad4.
Clinical significance
Role of Smad in cancer
Defects in Smad signaling can result in TGF-B resistance, causing dysregulation of cell growth. Deregulation of TGF-B signaling has been implicated in many cancer types, including pancreatic, colon, breast, lung, and prostate cancer. Smad4 is most commonly mutated in human cancers, particularly pancreatic and colon cancer. Smad4 is inactivated in nearly half of all pancreatic cancers. As a result, Smad4 was first termed Deleted in Pancreatic Cancer Locus 4 (DPC4) upon its discovery.
Germline Smad4 mutations are partially responsible for genetic disposition for human familial
juvenile polyposis
Juvenile polyposis syndrome is an autosomal dominant genetic condition characterized by the appearance of multiple juvenile polyps in the gastrointestinal tract. Polyps are abnormal growths arising from a mucous membrane. These usually begin appe ...
, which puts a person at high risk of developing potentially cancerous gastrointestinal
polyps. Experimental supporting evidence for this observation comes from a study showing that heterozygous Smad4 knockout mice (+/-) uniformly developed gastrointestinal polyps by 100 weeks. Many familial Smad4 mutants occur on the MH2 domain, which disrupts the protein’s ability to form
homo- or
hetero-oligomer
In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relativ ...
s, thus impairing TGF-B signal transduction.
Despite evidence showing that Smad3 is more critical than Smad2 in TGF-B signaling, the rate of Smad3 mutations in cancer is lower than that of Smad2.
Choriocarcinoma
Choriocarcinoma is a malignant, trophoblastic cancer, usually of the placenta. It is characterized by early hematogenous spread to the lungs. It belongs to the malignant end of the spectrum in gestational trophoblastic disease (GTD). It is also cl ...
tumor cells are TGF-B signaling resistant, as well as lacking Smad3 expression. Studies show that reintroducing Smad3 into choriocarcinoma cells is sufficient to increase TIMP-1 (tissue inhibitor of metalloprotease-1) levels, a mediator of TGF-B’s anti-invasive effect, and thus restore TGF-B signaling. However, reintroducing Smad3 was not sufficient to rescue the anti-invasive effect of TGF-B. This suggests that other signaling mechanisms in addition to Smad3 are defective in TGF-B resistant choriocarcinoma.
Role of Smad in Alzheimer’s
Alzheimer’s
Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As t ...
patients display elevated levels of TGF-B and phosphorylated Smad2 in their
hippocampal
The hippocampus (via Latin from Greek , 'seahorse') is a major component of the brain of humans and other vertebrates. Humans and other mammals have two hippocampi, one in each side of the brain. The hippocampus is part of the limbic system, an ...
neurons. This finding is seemingly paradoxical, as TGF-B has previously been shown to have neuroprotective effects on Alzheimer’s patients. This suggests that some aspect of TGF-B signaling is defective, causing TGF-B to lose its neuroprotective effects. Research has shown that phosphorylated Smad2 is ectopically localized to cytoplasmic granules rather than the nucleus, in hippocampal neurons of patients with Alzheimer’s disease. Specifically, the ectopically located phosphorylated Smad2s were found within
amyloid plaque
Amyloid plaques (also known as neuritic plaques, amyloid beta plaques or senile plaques) are extracellular deposits of the amyloid beta (Aβ) protein mainly in the grey matter of the brain. Degenerative neuronal elements and an abundance of mi ...
s, and attached to
neurofibrillary tangle
Neurofibrillary tangles (NFTs) are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary biomarker of Alzheimer's disease. Their presence is also found in numerous other diseases known as tauopathies. Little is kn ...
s. These data suggest that Smad2 is involved in the development of Alzheimer’s disease. Recent studies show that the
peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in promoting the abnormal localization of Smad2. Pin1 was found to co-localize with Smad2/3 and phosphorylated
tau protein
The tau proteins (abbreviated from tubulin associated unit) are a group of six highly soluble protein isoforms produced by alternative splicing from the gene ''MAPT'' (microtubule-associated protein tau). They have roles primarily in maintaining ...
s within the cytoplasmic granules, suggesting a possible interaction. Transfecting Smad2 expressing cells with Pin1 causes proteasome-mediated Smad2 degradation, as well as increased association of Smad2 with phosphorylated tau. This feedback loop is bidirectional; Smad2 is also capable of increasing Pin1 mRNA synthesis. Thus, the two proteins could be caught in a “vicious cycle” of regulation. Pin1 causes both itself and Smad2 to be associated in insoluble neurofibrillary tangles, resulting in low levels of both soluble proteins. Smad2 then promotes Pin1 RNA synthesis to try and compensate, which only drives more Smad2 degradation and association with neurofibrillary tangles.
TGF-β/Smad signaling in kidney disease
Dysregulation of TGF-B/Smad signaling is a possible pathogenic mechanism of
chronic kidney disease
Chronic kidney disease (CKD) is a type of kidney disease in which a gradual loss of kidney function occurs over a period of months to years. Initially generally no symptoms are seen, but later symptoms may include leg swelling, feeling tired, vo ...
. In the kidneys, TGF-B1 promotes accumulation of the
extracellular matrix
In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide stru ...
(ECM) by increasing its production and inhibiting its degradation, which is characteristic of renal
fibrosis
Fibrosis, also known as fibrotic scarring, is a pathological wound healing in which connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodelling and the formation of perma ...
. TGF-B1 signal is transduced by the R-Smads Smad2 and Smad3, both of which are found to be overexpressed in diseased kidneys. Smad3 knockout mice display reduced progression of renal fibrosis, suggesting its importance in regulating the disease. Conversely, inhibiting Smad2 in kidney cells (full Smad2 knockouts are embryonic lethal) actually leads to more severe fibrosis, suggesting that Smad2 works antagonistically to Smad3 in the progression of renal fibrosis. Unlike the R-Smads, Smad7 protein is typically under-expressed in diseased kidney cells. This loss of TGF-B inhibition results in increased amounts of active Smad2/3, which contribute to the progression of renal fibrosis as described above.
Notes
References
External links
*
{{TGFβ receptor superfamily modulators
Transcription factors
Protein families