User:Apolena Zounarová/Sandbox 2

NOTCH1 is a member of NOTCH family receptor proteins which consists of four members (NOTCH1-4). NOTCH proteins are evolutionarily highly conserved signalling proteins responsible for direct transduction of developmental signals at the cell surface into change in a transcriptional profile in the nucleus.

Structure and Function of Notch1

NOTCH receptors are class I transmembrane glycoproteins composed of an extracellular subunit and transmembrane and intracellular subunit, which interact via a specialised heterodimerization domain (HD). The extracellular subunit engages ligand via several EGF-like repeats and further contains three LIN-12/NOTCH repeats (LNR) which stabilise the dimerization domain by holding the two NOTCH subunits together. The transmembrane-intracellular subunit contains a short extracellular juxtamembrane peptide, transmembrane sequence and cytoplasmic domains including RAM domain, nuclear localization signals (NLS), a series of ankyrin repeats, glutamine-rich region (OPA) and C-terminal PEST domain which serves as a ligand-activated transcription factor ^[1].

Proteolytic Events During Notch1 Secretion and Signal Transduction

Furin-type Convertase Cleavage

Notch1 is posttranslationally modified by a proteolytic cleavage at S1 sites and reaches the plasma membrane as a heterodimer. Non-cleaved Notch1 is autoinhibited. Furin-type convertase is responsible for this process and cleaves Notch1 in at least two places: after R1633 and after R1664 ^[2]. Both residues are located in a loop exposed into the cytosol and lie approximately 100 and 70 amino acids external from the transmembrane region, respectively ^[2][3].

Additional Cleavages in Response to Receptor Activation

Binding of Notch1 ligands such as Delta1 or Jagged1 leads to the dissociation of the heterodimer. This structural change reveals S2 site for a cleavage by metalloprotease TNAα-converting enzyme (TACE), a member of a disintegrin and metalloprotease domain (ADAM) family, which then produces a fragment termed as Notch extracellular truncation (NEXT). S2 site is located between A1710 and V1711 in murine Notch1, 13 amino acids from the TM domain ^[4][5], which corresponds with positions 1720 and 1721 in human Notch1 ^[3]. The mechanism of ligand-induced dissociation can be explained by mechanical force caused by simultaneous endocytosis in the ligand cell. Thus, the events in the ligand cell are important for the Notch signal transduction as well. Since S2 cleavage is a ligand-regulated step, mutations in heterodimerization domain can mimic ligand-bound stage of the receptor and facilitate Notch proteolysis in a similar manner ^[6]. The cleavage by metalloprotease probably brings the receptor in a conformation similar to that of constitutively active receptors ^[4]. Although the cleavage at S2 site is prominent for the activation of the Notch pathway ^[4], subsequent cleavage by γ-secretase presenilin at S3 site is the one which is responsible for Notch intracellular domain (NICD) production ^[7]. γ-secretase cleaves between G1743 and V1744 in murine Notch1 ^[8], which is G1753 and V1754 in human Notch1 ^[3]. The same enzymatic activity creates Aβ peptide in Alzheimer‘s disease from β-APP precursor ^[7].

Heterodimerization Domain and Negative Regulatory Region

Stable association of the two Notch1 chains depends on the heterodimerization domain which consists of two regions. 65 amino acid C-terminal region (HD-C, NTM) remains associated with the transmembrane part of the receptor, whereas 103 aminoacid extracellular N-terminal region (HD-N, NEC) has been separated by furin-type convertase and interacts with the membrane-bound part non-covalently. Adjacent to HD-N are three LIN-12/NOTCH repeats (LNR) which are not necessary for heterodimerization, but rather protect HD-C from metalloprotease cleavage and prevent ligand-independent activation of the signalling pathway. LNR are together with HD-N termed as the negative regulatory region, NRR ^[9]. Notch mutants with deleted extracellular domain are constitutively active and are cleaved at the S3 site in a constitutive manner. Also, their activity is equivalent to Notch mutants containing the intracellular part only. This observation supports the idea that the conformation of the receptor is important for the changes in accessibility of S3 site. Ligand binding can change the conformation and permits cleavage of Notch by γ-secretase either directly or indirectly. On the contrary, constructs bearing also LNR are neither constitutively active nor constitutively cleaved ^[10].

T-cell Acute Lymphoblastic Leukaemia

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Structural highlights

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