Chaperones

Chaperones are a functionally related group of proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures.

1. CHAPERONES

2. Chaperones are a functionally related group of proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures F ound in all types of cells and cellular compartments, and have a wide range of binding specificities and functional roles. bind non native proteins preventing unspecific aggregation What are Chaperones?

3. A landmark feature of molecular chaperones is the involvement of energy-dependent reactions in the folding process The misfolded or unfolded polypeptide chains to which chaperones bind are said to be "non-native," as they are not folded into their functional conformation

4. The molecular chaperones are a diverse group of families of proteins that are requires for the C orrect folding T ransport D egradation of others proteins in vivo They are formed from amino acid monomers They do not interact with native proteins, nor do they form part of the final folded structures .

5. Chaperones are present when the macromolecules Perform their normal biological functions Correctly completed the processes of folding and/or assembly The chaperones are concerned primarily with protein folding Some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specific targets.

6. Location In humans mostly in Endoplasmic reticulum General chaperones: GRP78/ BiP , GRP94, GRP170. Lectin chaperones: calnexin and calreticulin Non-classical molecular chaperones: HSP47 and ERp29 Folding chaperones: Protein disulfide isomerase (PDI ) Peptidyl prolyl cis -trans- isomerase (PPI ) ERp57

7. The term ` molecular chaperone ` appeared first in the literature in 1978 Invented by Ron Laskey History T o describe the ability of a nuclear protein called ‘ Nucleoplasmin ’ It prevented the aggregation of folded histone proteins with DNA during the assembly of nucleosomes

8. Later extended by R. John Ellis in 1987 to describe proteins that mediated the post-translational assembly of protein complexes . In 1988, realised that similar proteins mediated this process in both prokaryotes and eukaryotes. D etails were determined in 1989, when the ATP-dependent protein folding was demonstrated in vitro.

9. Properties of chaperones Molecular chaperones interact with unfolded or partially folded protein subunits e.g. nascent chains emerging from the ribosome, or extended chains being translocated across subcellular membranes. They stabilize non-native conformation and facilitate correct folding of protein subunits. They do not interact with native proteins, nor do they form part of the final folded structures.

10. Some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specific targets. They often couple ATP binding/hydrolysis to the folding process. Essential for viability, their expression is often increased by cellular stress.

11. As heat shock proteins Heat shock proteins (HSP) are a family of proteins that are produced by cells in response to exposure to stressful conditions Several heat shock proteins function as intra-cellular chaperones for other proteins In bacteria like E. coli, chaperones are highly expressed under high stress e.g high temperatures.

12. For this reason, the term "heat shock protein" has historically been used to name these chaperones. The prefix " Hsp " designates that the protein is a heat shock protein. The reason for this behaviour is that protein folding is severely affected by heat and therefore, some chaperones act to prevent or correct damage caused by misfolding.

13. Functions of chaperones They act as a container for the folding of other protein sub units as they are called heat-shocked proteins. They also prevent the degradation of proteins in spinal stress conditions. Some chaperone systems work as foldases . They support the folding of proteins in an ATP-dependent manner .(for example, in the GrpE or the DnaK / DnaJ / GrpE system).

14. Other chaperones work as holdases . They bind folding intermediates to prevent their aggregation, for example DnaJ or Hsp33. Chaperones work in coordination by forming assemblies . Such assembly chaperones, especially in the nucleus are concerned with the assembly of folded subunits into oligomeric structures. The first protein to be called a chaperone assists the assembly of nucleosomes from folded histones and DNA .

15. They recognize and correct mistakes in folding by binding to the non polar surface. Promote correct folding of their substrate proteins by unfolding incorrect polypeptide chain conformations. Providing an environment in which correct protein folding can occur.

16. Macromolecular crowding The crowded environment of the cytosol can accelerate the folding process. As a compact folded protein will occupy less volume than an unfolded protein chain. Crowding can reduce the yield of correctly folded protein by increasing protein aggregation. Crowding may also increase the effectiveness of the chaperone proteins such as Grp E which could counteract this reduction in folding efficiency.

17. Molecular chaperones of bacteria behave as direct virulence factors. Effects of bacterial molecular chaperones on host cells include cell-cell signalling and promoting apoptosis

18. Cell homeostasis Two opposite functions Protein folding Degradation. The two processes Are carried out through the transient formation of complexes Between different chaperones and co-chaperones

19. Translocation Transport across membranes Across membranes of the mitochondria E ndoplasmic reticulum (ER). Bacterial translocation—specific chaperone maintains newly synthesized precursor polypeptide chains in a translocation-competent state and guides them to the translocon .

20. New functions for chaperones continue to be discovered, such as Assistance in protein degradation Bacterial adhesion activity In responding to diseases linked to protein aggregation and cancer maintenance.

21. They prevent inappropriate association or aggregation of exposed hydrophobic surfaces Direct their substrates into productive folding Transport or degradation pathways Main Role

22. The type I interamolecular chaperones First discovered based on the studies on subtilisin , an alkaline serine protease from bacillus subtilis . Mediate the folding of proteins into their respective tertiary structures and are mostly produced as the N-terminal sequence extension.

23. Mediate the formation of the quaternary or functional structure of proteins Usually located at the C-terminus of the protein Type II intramolecular chaperones

24. Mutations in the intramolecular chaperones can cause misfolding of the functional domain, results in distortion of their function leading to human diseases.

25. Families Many families present in eukaryotes and prokaryotes Perform many similar and specific functions by working in coordination systems Most common are hsp 70 and hsp 60 families

26. Small heat shock proteins (hsp25) [holders] Hsp25 is the second largest of 16 identifiable small heat shock proteins in the nematode. Protect against cellular stress Prevent aggregation in the lens (cataract) Hsp90 ATPase [holder] Hsp90 is a specialized chaperone that assists in the maturation of client proteins. These proteins include over a hundred transcription factors and kinases, such as steroid receptors

27. Hsp90 Hsp90 ( HtpG in E. coli) may be the least understood chaperone The exact function of Hsp90 is also currently a mystery. Researchers don't know what it does in the maturation of its client proteins. They have discovered that it acts as part of a large complex of different chaperone proteins. Some of these chaperones deliver immature proteins to the complex, and others assist with folding. essential for activating many signalling proteins in the eukaryotic cell Each Hsp90 has an ATP-binding domain, a middle domain, and a dimerization domain

28. Hsp90 (blue) and cochaperone Sba1 (green), with bound ATP (red).

29. Calnexin , calreticulin Calnexin (CNX) is a 67kDa integral protein of the endoplasmic reticulum (ER) Calreticulin also known as calregulin in humans is encoded by the CALR gene . Calreticulin is a multifunctional protein that binds Ca2+ ion rendering it inactive

30. Hsp100 ( Clp ) ATPase [ unfolder ] The HSP100/ Clp proteins are a newly discovered family, promotion of proteolysis of specific cellular substrates and regulation of transcription. Common ability is to disassemble higher-order protein structures

31. Hsp100 Hsp100 ( Clp family in E. coli) proteins have been studied in vivo and in vitro Ability to target and unfold tagged and misfolded proteins. Form large hexameric structures Unfoldase activity in the presence of ATP .

32. Proteins in the Hsp100/ Clp family form large hexameric structures with unfoldase activity in the presence of ATP. These proteins are thought to function as chaperones by processively threading client proteins through a small 20 Å (2 nm) pore Gives each client protein a second chance to fold. Forms complexes that are responsible for the targeted destruction of tagged and misfolded proteins.

33. Hsp 104 Hsp104 = the Hsp100 of Saccharomyces cerevisiae Essential for the propagation of many yeast prions. Deletion of the HSP104 gene results in cells that are unable to propagate certain prions.

34. Hsp70 chaperones Their size is approximately 70,000 daltons B est characterized small (~ 70 kDa ) chaperone Often work in concert with one or more smaller co-chaperone proteins , which serve to modulate the activity of the chaperone The Hsp70 proteins are aided by Hsp40 proteins ( DnaJ in E. coli), which increase the ATP consumption rate and activity of the Hsp70s

35. Include DnaK from the bacterium Escherichia co li The Ssa and Ssb proteins from yeast BiP ( for "binding protein") from the mammalian endoplasmic reticulum

36. Hsp70 consists of ATP-binding N-terminal domain and peptide binding C-terminal domain. ATP hydrolysis switches off and on the binding ability of C-terminal domain. A special hydrophobic groove formed by α- helices and β- strands provides the docking site F or hydrophobic segments of misfolded proteins

37. Hsp70s crowd around an unfolded substrate, stabilizing it and preventing aggregation Until the unfolded molecule folds properly, at which time the Hsp70s lose affinity for the molecule and diffuse away Hsp70 also acts as a mitochondrial and chloroplastic molecular chaperone in eukaryotes I ncreased expression of Hsp70 proteins in the cell results in a decreased tendency toward apoptosis.

38. Hsp 60 also called " chaperonins " are barrel-shaped structures Composed of fourteen to sixteen subunits of proteins that are approximately 60,000 daltons in size The best characterized large (~ 1 MDa ) chaperone complex

39. Each subunit has a patch of non-polar amino acid groups lining the inner surface of the barrel T his patch recognizes the exposed non-polar amino acids of misfolded proteins. The binding and hydrolysis of ATP triggers conformational changes within the barrel

40. Most extensively studied Hsp60 chaperones include GroEL and GroES from E. coli TRiC /CCT from eukaryotic cells TRiC /CCT recognizes a much smaller set of proteins, and appears to play an additional role in the assembly of multiprotein complexes

41. GroEL is a double-ring 14mer with a hydrophobic patch at its opening GroES is a single-ring heptamer that binds to GroEL in the presence of ATP or ADP GroEL and GroES forms a well understood complex

42. GroEL and GroES complex GroEL chaperone consists of two rings – cis (or proximal) upper ring and trans (or distal)lower ring. GroES co-chaperone binds to both GroES rings Each of the GroEL rings consists of seven identical units shown in the lower The units are arranged in a circular manner and form a cavity GroES upon binding to GroEL serves as “lid”, which encapsulates the volume inside the cavity

43. Complete their cycle in 4 phases Capture (T state) ATP hydrolysis (R’’ state) Encapsulation Substrate release

44. A top-view of the GroES / GroEL bacterial chaperone complex model

45. Chaperones and Human Disease It is clear that molecular chaperones assist with the folding of newly synthesized proteins and correct protein misfolding. Recent studies now suggest that defects in molecular chaperone/substrate interactions may also play a substantial role in human disease

46. For example Mutations linked to Alzheimer's disease have been shown to disrupt the expression of chaperones in the endoplasmic reticulum Several genes linked to eye degeneration diseases have recently been identified as putative molecular chaperones