Hedgehog signalling pathway

The Hedgehog proteins were discovered in Drosophila .
 At least three genes encode Hedgehog proteins in vertebrates—Sonic, Desert, and Indian hedgehog. The active forms of all Hedgehog proteins are covalently coupled to cholesterol, as well as to a fatty acid chain.

 The effects of Hedgehog are mediated by a latent transcription regulator called Cubitus interruptus (Ci).
1.In the absence of a Hedgehog signal- Most Patched is in intracellular vesicles , where it keeps Smoothened inactive and sequestered. The Ci protein is bound in a cytosolic protein degradation complex, which includes the protein kinase Fused and the scaffold protein Costal2. Costal2 recruits three other protein kinases (PKA, GSK3, and CK1; not shown), which phosphorylate Ci. Phosphorylated Ci is ubiquitylated and then cleaved in proteasomes  to form a transcriptional repressor, which accumulates in the nucleus to help keep Hedgehog target genes inactive.

 2.In the presence of a Hedgehog  signal-  Binding to iHog and Patched removes the inhibition of Smoothened by Patched. Smoothened is phosphorylated by PKA and CK1 and translocates to the plasma membrane, where it recruits the complex containing Fused, Costal2, and Ci. Costal2 releases unprocessed Ci, which accumulates in the nucleus and activates the transcription of Hedgehog target genes. Many details in the pathway are poorly understood, including the role of Fused.

Hedgehog functions by blocking the proteolytic processing of Ci, thereby changing it into a transcriptional activator. It does this by a convoluted signaling process that depends on three transmembrane proteins:
                                       ( Patched, iHog, and Smoothened. )

. Hedgehog signaling can promote cell proliferation, and excessive Hedgehog signaling can lead to cancer. Inactivating mutations in one of the two human Patched genes, for example, which lead to excessive Hedgehog signaling, occur frequently in basal cell carcinoma of the skin, the most common form of cancer in Caucasians. A small molecule called cyclopamine, made by a meadow lily, is being used to treat cancers associated with excessive Hedgehog signaling. It blocks Hedgehog signaling by binding tightly to Smoothened and inhibiting its activity.

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The PI-3-Kinase –Akt Signaling Pathway

Extracellular signals are usually required for animal cells to grow and divide, as well as to survive .

Members of the insulinlike growth factor (IGF) family of signal proteins, for example, RTKs stimulate many types of animal cells to survive and grow. 

1.They bind to specific RTKs which activate PI 3-kinase to produce PI(3,4,5)P3. 
2.The PI(3,4,5)P3 recruits two protein kinases to the plasma membrane via their PH domains—
                 a. Akt (also called protein kinase B, or PKB) 
                 b. Phosphoinositide-dependent protein kinase 1 (PDK1), and this leads to the activation of Akt ).
3. Once activated, Akt phosphorylates various target proteins at the plasma membrane, as well as in the cytosol and nucleus. 
4.Actions of Akt all conspire to enhance cell survival and growth.

The control of cell growth by the PI-3-kinase–Akt pathway depends in part on a large protein kinase called TOR (named as the target of rapamycin, a bacterial toxin that inactivates the kinase and is used clinically as both an immunosuppressant and anticancer drug). 

TOR was originally identified in yeasts in genetic screens for rapamycin resistance; in mammalian cells, it is called mTOR, which exists in cells in two functionally distinct multiprotein complexes. 

1.  mTOR complex 1- Contains the protein raptor; this complex is sensitive to rapamycin, and it stimulates cell growth—both by promoting ribosome production and protein synthesis and by inhibiting protein degradation. 

Complex 1 also promotes both cell growth and cell survival by stimulating nutrient uptake and metabolism. The mTOR in complex 1 integrates inputs from various sources, including extracellular signal proteins referred to as growth factors and nutrients such as amino acids, both of which help activate mTOR and promote cell growth. The growth factors activate mTOR mainly via the PI-3-kinase–Akt pathway. Akt activates mTOR in complex 1 indirectly by phosphorylating, and thereby inhibiting, a GAP called Tsc2. Tsc2 acts on a monomeric Ras-related GTPase called Rheb . Rheb in its active form (Rheb-GTP) activates mTOR in complex 1. The net result is that Akt activates mTOR and thereby promotes cell growth 

2.mTOR complex 2 -Contains the protein rictor and is insensitive to rapamycin; it helps to activate Akt , and it regulates the actin cytoskeleton via Rho family GTPases. 

The Akt is phosphorylated on a serine by a third kinase (usually mTOR in complex 2), which alters the conformation of the Akt so that it can be phosphorylated on a threonine by PDK1, which activates the Akt. The activated Akt now dissociates from the plasma membrane and phosphorylates various target proteins, including the Bad protein. When unphosphorylated, Bad holds one or more apoptosis-inhibitory proteins (of the Bcl2 family—) in an inactive state. Once phosphorylated, Bad releases the inhibitory proteins, which now can block apoptosis and thereby promote cell survival. 

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UNIT 1 BIOCHEMISTRY CSIR NET ANFINSEN'S EXPERIMENT

 The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds 

 Anfinsen's plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure.

AGENTS -

1. Urea or guanidinium chloride-   Effectively disrupt the noncovalent bonds.
2.β-mercaptoethanol   -The disulfide bonds can be cleaved reversibly by reducing them with a reagent such as β-mercaptoethanol . In the presence of a large excess of β-mercaptoethanol, a protein is produced in which the disulfides (cystines) are fully converted into sulfhydryls (cysteines).

EXPERIMENT-

A. When ribonuclease was treated with β-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment.

B. Anfinsen then made the critical observation that the denatured ribonuclease, freed of urea and β-mercaptoethanol by dialysis, slowly regained enzymatic activity . The sulfhydryl groups of the denatured enzyme became oxidized by air, and the enzyme spontaneously refolded into a catalytically active form. Detailed studies then showed that nearly all the original enzymatic activity was regained if the sulfhydryl groups were oxidized under suitable conditions. 

C. A quite different result was obtained when reduced ribonuclease was reoxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1% of the enzymatic activity of the native protein. 

 The reason is that the wrong disulfides formed pairs in urea. There are 105 different ways of pairing eight cysteine molecules to form four disulfides; only one of these combinations is enzymatically active. The 104 wrong pairings have been picturesquely termed "scrambled" ribonuclease. 

D.Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of β-mercaptoethanol were added to an aqueous solution of the protein  The added β-mercaptoethanol catalyzed the rearrangement of disulfide pairings until the native structure was regained in about 10 hours. This process was driven by the decrease in free energy as the scrambled conformations were converted into the stable, native conformation of the enzyme. The native disulfide pairings of ribonuclease thus contribute to the stabilization of the thermodynamically preferred structure.

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