in vivo proteon folding

8/2/2019 In Vivo Proteon Folding

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MOLECULAR CHAPERONES

AND

IN-VIVO PROTEIN FOLDING

Prepared by-

Bhishma Patel

209BM2246

M.Tech Biotech (1st sem)


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INTRODUCTION

Protein folding is the physical process by which apolypeptide folds into its characteristic andfunctional three dimensional structure from randomcoil

Each amino acid has to some extent a specialcharacter, which determines more or less theposition of the amino acid residue in the nativeprotein, and therefore determines the overall protein

structureC. Anfinsion postulated that all proteins contains

in their primary structure, the complete informationwhich determines the 20 & 30structure


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INVITROVS.INVIVOFOLDING

folding by dilution

in buffer

protein denatured

in a chaotrope

folded

protein

in vitro in vivo

folding

folded

protein

Differences:

1. One has all of the

information

immlediately available

for folding; the other

process is gradual

2. the cellular

environment is very

different (much more

crowded)


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INVITROVS.INVIVOFOLDING

The environment in which the protein folding occurs

within cell.

- The concentration of unfolded & nacent chain in the

cytoplasm & in the context of ribosomes very high. Formation of tertiary structure requires the presence

of complete polypeptide or at least a complete

protein domain.

After folding, protein must fulfill their function in anenvironment in which fluctuation in temperature and

chemical composition occurs


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intramolecular

misfolding

X

X

X

X

intermolecular

aggregation

X

X

X

X

X

X

Incorrectmolecular

interactions

&loss of activity

exposed

hydrophobic

residues

CONTD

Non-native proteins expose hydrophobic residues that are

normally buried within the core of the proteinThese hydrophobic amino acids have a strong tendency

to interact with other hydrophobic (apolar) residues -especially under crowding conditions


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MOLECULARCHAPERONES

Molecular chaperones are protein machines thatrecognize non-native states of other proteins and, bycontrolled binding and release, assist these substrateproteins to fold properly.

In the late 1970s, the term Molecular chaperone wascoined to describe the properties of nucleoplasmin:

Nucleoplasmin prevents incorrect interactions betweenhistones and DNA

In the late 1980s, the term molecular chaperone was

used more broadly by John Ellis to describe the roles of

various cellular proteins in protein folding and assembly


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Requirements for a protein to be considered a

chaperone:

(1) interacts with and stabilizes non-native forms of

protein(s)

- technically also: folded forms that adopt different

protein conformations

(2) not part of the final assembly of protein(s) means it

act as catalysts


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NEEDSOF MOLECULARCHAPERONES

To prevent the misfolding and aggregation during the

folding of newly synthasized chains

To prevent nonproductive interaction with other cell

component

To direct the assembly of larger protein and multiprotein

complexes

During stress condition, help in refolding of denatured

proteins

Assisting in the process of proteolytic degradation


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MOLECULARCHAPERONESINVOLVEDIN

IN-VIVOPROTEINFOLDING

Small Heat Shock Proteins

Hsp 40 and DnaJ Family

Nascent polypeptide associated-complex (NAC)

Hsp 60 Family Hsp 70 Family

Hsp 90 Family

Hsp100 Family

Protein Disulfide Isomerase

Peptidyl Prolyl Isomerase / Trigger Factor

Specialized chaperones


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Folding pathway for a

model protein

involving three

chaperone

Systems in the bacterial

cytosol


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SMALL HEAT SHOCK PROTEINS

Consist of 12-43 KDa proteins that assembles into large

multimeric structure.

They prevent protein aggregation in an ATP dependent

manner and also to some extent solubilize aggregates.

Little is known about mechanism of action. It has been

suggested that the substrate protein coats the outside of

the large chaperone multimer and that hydrophobic

interactions are critical in substrate binding

e.g -crystallins found in our eye lenses where its major

role is to bind denatured protein and prevent their

aggregation.


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DNAJ/HSP40 FAMILY

It consist of 100 menber, defined by presence of highly

conserved J domain of 78 residues.

J domain is a motif of 4 -helices with conserved

sequence HPD in loop between helix 2&3 which

followed by nonconserved C terminus

It require for efficient binding of protein to Hsp70

through simulation of its ATPase activity

It can directly interact with denatured substrate proteins

DnaJ/Hsp40 has been proposed to bind with nascent

polypeptides to prevent premature folding and to target

Hsp70 to them.


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HSP 90 FAMILY

Highly conserved and essential protein found in all

organism from bacteria to human

e.g in Euk. Hsp 90, the ER form Grp94 and the

E.coli homolog HtpG. It has some specific interaction, e.g. with

cytoskeleton elements, signal transduction proteins

and protein kinase.

In vivo function are poorly understood.

It functions in association with other cofactors like,

PPI family, FKBP52, p23 and steroid recepter

complex consist of Hsp90, Hsp70, p48 etc..


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NASCENTPOLYPEPTIDEASSOCIATED-

COMPLEX (NAC)

It is heterodimer of 21KDa & 33KDa subunit

It binds to nascent chain at ribosome exit site

It prevent the association of ribosome with protein

translocation machinery of the ER membrane. It involved in the targeting pre proteins to different

sub cellular location such as ER and mitochondria

Also cooperate with the Hsp70 system in preventing

early folding and aggregation of protein


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HSP 70 FAMILY

It is very large family of molecular chaperonesinvolved in protein folding, with multiplemembers present in most organism.

e.g. Hsc70

constitutive cytosolic memberHsp70 the stress induced cytosolic form

BiPthe ER form

mHsp70 the mitochondrial form

DnaK prok. Equivalent of Hsc70 foundalso in mitochandria and plastid

Ssa1-4 & Kar2 the homologs of Hsp70 &BiP in yeast respectively.


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CONTD

They have two primary domains:

an ATPase domain

a peptide-binding domain

an ATPase domain consist of four smaller domainsforming two lobes with a deep cleft within which the

MgATP & MgADP bind.

a peptide-binding domain bind to segments of unfolded

polypeptides, particularly those containing hydrophobicresidues, and release them in an ATP-dependent manner


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FUNCTIONOF HSP70/DNAK

DnaK, in cooperation with DnaJ, binds to exposed

hydrophobic segments of the nascent polypeptide chain

and prevent misfolding or aggregation.

Associated with proteins that are translocated into the

lumen of the endoplasmic reticulum in a co-translational

manner and prevents misfolding or aggregation

In the case of mitochondria, unfolded pre-proteins are

generally transported post-translationally across both

membranes into the matrix, where they interact with anHsp70 that facilitates both their translocation and folding


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DNAK REACTIONCYCLE


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HSP70 COCHAPERONES

GrpE

found in bacteria and mitochondria and facilitates

nucleotide release from Hsp70

Detailed mechanism is unclear

Hip

It stabilise the ADP state of Hsc70 that has a high affinity

for substrate protein - forming stable Hsp70 complexwith substrate proteins

it also binds to some unfolded protein.


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BAG-1

Is an antiapoptotic protein and also interact with several

steroid hormone recepters

It binding to the ATPase domain, stimulate the rate ofATP hydrolysis by increasing the rate of release of ADP

from Hsp70

P16 It modulates the Hsc70 function by maintaining Hsc70 in

a monomeric state and by dissociating unfolded protein

from Hsc70


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Auxilin

100KDa cofactor involved in the Hsp70 mediateduncoating of clathrin-coated vesicles.

It binds to assembeled clathrin lattices and in the

presence of ATP , recruits Hsp70. The presence of J domain at COOH terminus indicates

its a member of DnaJ family

Hop

60KDa protein that can form physical link betweenHsp70 and Hsp90

It involved in the refolding of denatured protein in rabbitreticulocyte lysate


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HSP60 FAMILYORCHAPERONIN

They assemble into large, double ring structures and,

together with the co-chaperonin known as Hsp10 or

GroES, provide a central cavity that allows proteins of

size up to about 6065 kDa to fold in a protected

environment.

e.g. GroELin prokarotes, mitochondria and

chloroplast

TCP-1 ring complex (TRiC) in eukaryotes

chaperonins was originally coined by Ellis to refer non

heat induced Hsp60


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FUNCTION

GroEL is most studied

It facilitates protein folding by preventing

aggregation and also allow partially folded

intermediates to fold in an environmentconducive to stabilizing the native state

It also function by unfolding the misfolded state

so as to allow their productive folding.

Member of Hsp60 family also involved in the

assembly of large multiprotein complex.


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STRUCTUREOF HSP60/GROEL

It consist of 14 identical subunits in two stackedheptameric rings, each containing central cavity.

GroEL subunit consist of three domains:

Equatorial contains nucleotides binding site

Intermediate binds substrate protein

Apical binds GroES

In Euk. Similar complex called TRiC which is hetero

oligomer of 8 different subunits. In thermophillic archea, the chaperonin is a

homooctamer with build in lid, for stability againstthermal dissociation.


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HSP60 REACTIONCYCLE


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HSP100 FAMILY

Contain ATP and polypeptide binding domains

Both Hsp104 and Clp form six membered ring

complex

No human analogs of Hsp104 have been found

It may act in concert with Hsp70 and DnaJ

homologs to increase the yields of renatured

protein

Hsp104 has been observed to solubilize

thermally aggregated proteins both in vivo and

in vitro


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PREOTEIN DISULFIDE ISOMERASE (PDI)

It is a widely distributed enzyme that catalyzesthe interchange or shuffling of disulfide bondsuntil the bonds of the native conformation areformed.

S-S bond formation occurs rapidly and isfollowed by thiol disulfide rearrangementleading to the correct S-S pairing.

It also binds relatively hydrophobic moleculessuch as steroid and thyroid hormones.

It has two catalytic sites, one near to the NH2terminus and other near to COOH terminus.


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PEPTIDYL PROLYL ISOMERASE (PPI)

PPI catalyzes the interconversion of the cis and trans

isomers of Pro peptide bonds (Fig. 48b), which can be a

slow step in the folding of proteins that contain some Pro

residue peptide bonds in the cis conformation.

Three unrelated families are known:

the cyclophilins

FK506-binding protein (FKBP)

parvulins


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TRIGGER FACTOR

48 kDa protein which was first identified by its ability tomaintain the precursor of a secretory protein in atranslocation competent form in E.coli

Trigger factor has three domains:

an amino-terminal ribosome-binding domain a middle domain with prolyl isomerase activity

a carboxy-terminal domain with no function has beenclearly defined

It binds to nascent cytosolic and secretory polypeptidechain and catalyze protein folding in vitro.

GroEL-TF complex show much greater affinity forpartially folded intermediate than GroEL alone


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SPECIALIZEDCHAPERONES

PapDinvolved in the assembly of bacterial pili.

Hsp47

Found in collagen producing cells

Involved in the folding and processing of procolagen inthe ER.

SecB-

Found in E. coli has two function: it maintain precursorof some exported protein by preventing their aggregation

or folding to their native state in cytoplasm and it

delivers both nascent and completed precursor to SecA.


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PROTEINFLUXTHROUGHBACTERIAL

CHAPERONESYSTEMS


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MITOCHONDRIALIMPORT/FOLDING

Molecular chaperones play critical role in targeting

protein to the mitochondria and the subsequent folding of

the imported protein.

Two different mHsp70 complexes-

The ADP bound form favors formation of a complex on

the inner membrane that contains mHsp70, its membrane

anchor Tim44 and mGrpE.

The ATP bound form favors the frmation of a folding

complex in the matrix that contains mHsp70, the

mitochondrial DnaJ homolog Mdj1 and mGrpE.


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FOLDINGIN ER

Folding begins with the insertion of a preprotein into the

lumen of the ER and can occur either posttranslationally

or cotranslationally

ER has excellent quilty control mechanism that

selectively retain misfolded protein which are either

degraded or refolded.

Several proteins have been identified which are involved

in folding in ER BiP, Hsp90, calreticulin, three member

of thioredoxin superfamily: PDI, ERp72 and p50.


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CONCLUSION

Molecular chaperones recognize and bind to nascent

polypeptide chains and partially folded intermediates of

proteins, preventing their aggregation and misfolding.

Wide variety of techniques ranging from genetics to

biophysics have begun to unravel the complexities of

these chaperone machines.

Different cellular locatons, with their different role in

production of new proteins, have specific chaperone

systems tailored to the demand of the specific location.


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THANK YOU

in vivo proteon folding

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