CHAPTER I

Heat shock proteins and a-crystallin

1.1 HEAT-SHOCK PROTEINS (HSPs)

Prokaryotes and eukaryotes respond to sudden increases in temperature by synthesising a small

set of proteins called the heat shock proteins. This response has been highly conserved

throughout evolution, not only as a physiological phenomenon, but also at the level of individual

proteins. These proteins not only allow cells to recover from the stress, but also help in surviving

subsequent stronger stress. This phenomenon was first realised in 1974 when Drosophila cells

were subjected to heat shock (Tissieres et al., 1974). Since elevated temperature was the first

well-characterised stress system, a majority of these·proteins are referred to as heat shock

proteins. Subsequently, it was realised that, besides elevated temperature, the term stress could

be used in a very broad sense to describe osmotic stress, presence of ethanol, metabolic poisons

such as arsenite, drugs like puromycin, amino acid analogues, calcium ionophores or oxidants

in the external growth medium. Stress may also be induced when the cells are subjected to

glucose starvation, ischemia or pathogens. Under all these conditions, the levels of heat shock

proteins are elevated.

Investigations on the stress response at the molecular level were essentially concentrated on the

mechanisms that caused the mRNA transcripts of the stress proteins to accumulate. This system

provides an excellent example to study inducible gene expression. The sequences responsible for

heat shock gene expression, referred to as the heat shock element, are conserved from yeast to

humans (Kingston, 1990). This element consists of an inverted repeat sequence of about 5

nucleotide base pairs, nGAAn (Perisic et al., 1990) and is located 80-150 base pairs upstream of

the start site of RNA transcription. Its presence is the most definitive indication that the gene

codes for a heat shock protein. However, not much was known for a very long time as to how

6

these proteins helped the cells to overcome thestress, how these stress signals were transmitted

to the nucleus and how the cells sensed the stress per se.

The cellular functions of these diverse groups of stress proteins were not clearly understood as

different environmental treatments induced the same set of stress proteins. It was later realised

that these different environmental stresses lead to production of misfolded proteins in the cell

(Morimoto et al., 1990). Supporting evidence came from studies, which showed an increase in

the levels of these stress proteins in E. coli when there was an overproduction of heterologous

proteins, which quite often led to an increase in the levels of misfolded proteins in the cell. Stress

proteins were also induced by regulatory elements, which were distinct from the heat shock

element, which was the only regulatory element that was discovered till then. These distinct

regulatory elements could be triggered by protein factors like the E1A protein of the adenovirus,

heavy metals, serum factors etc (Morimoto et al., 1990). Some of the heat shock proteins (Hsps)

are expressed at basal levels in cells even under normal conditions of growth. One of the most

obvious interpretations of these results is that the HSPs are synthesised for reasons other than

heat shock.

What are the additional functions that heat shock proteins perform besides conferring

thermotolerance to cells? Considerable stimulus was given to this area of research when it was

proposed that some of these heat shock proteins could function as molecular chaperones and

assist proteins to assemble or disassemble (Pelham, 1986).

7

1.2 MOLECULAR CHAPERONES

Molecular chaperones can be described as proteins that bind to and stabilise an otherwise

unstable conformer of another protein, and, by controlled binding and release of the substrate

protein, facilitate its correct fate in the cell. A chaperone would thus facilitate folding, correct

oligomeric assembly, transport from one compartment in the cell to another, degradation, and/or

control the switching of a molecule from active to inactive state or vice versa by binding to it.

Stress proteins appear to perform most of these functions. The requirement for stabilising

proteins increases when proteins start to denature under stress. To understand the function of

molecular chaperones, it is essential to look at the cellular environment in totality. The cellular

environment is highly crowded because of the presence of high concentrations of proteins and

RNA. The concentration is of the order of 200-400 mg/ml of protein and RNA (Zimmerman and

Minton, 1993). This leads to an increase in the thermodynamic activities of the macromolecules

by several orders of magnitude. This effect is simply because the concentration of

macromolecules is very high and a significant volume of the cell would be unavailable to the

interacting species. Thus, association constants for two molecules that are interacting within the

cell are far greater than those measured for the same proteins in a test tube, where the

concentrations of the interacting species might not increase higher than 1 mg/ml. Macromolecular

crowding would increase the propensity of proteins to aggregate non-specifically during or

immediately after synthesis. Molecular chaperones would thus play a vital role in preventing this

non-specific aggregation and subsequently facilitate the correct folding by either providing a

secluded space to fold or by controlled release of these partially folded structures, thereby

enhancing the possibility of correct folding (Craig et al., 1993). In the light of the present

discussions, it is very important to delineate the differences between HSPs and molecular

8

chaperones. Not all the chaperones are heat shock proteins and not all the heat shock proteins can

act as chaperones. For example, the first molecular chaperone characterised, nucleoplasmin,

which assists in the folding of the histone-DNA complex, is not heat-inducible. Likewise only

5 of the 20 proteins regulated by the stress transcriptional activator sigma 32 are known to

function as molecular chaperones in vitro (Georgopoulos et al., 1990).

1.3 CLASSIFICATION OF THE HSPs

Since there exist a large number of heat shock proteins: a convenient nomenclature was adopted

based on the migration of these proteins during electrophoresis on sodium dodecyl sulphate

polyacrylamide gels. The HSPs can be broadly classified into 5 major groups; namely HSP100

(MR 100 kDa and above), HSP90, HSP70, HSP60 and the small HSPs (12-43 kDa). All these

groups contain conserved protein families and the numbers represent rounded-off values for the

apparent relative molecular masses of their subunits. In this section, a brief introduction to the

family of HSPs is presented.

1.3.1 HSPlOO family of proteins

Most eukaryotes produce proteins greater than 100 kDa in response to high temperature. These

have been characterised to some extent only in the case of mammals and yeasts. The 110 kDa

protein in murine cells is found in the nucleus. When the cells are given a heat shock they tend

to form a ring-like structure around the nucleolar periphery (Welch and Suhan, 1986).

Immunoelectron microscopy demonstrates that this protein associates with the fibrillar

component of the nucleoli. Treatment of the fixed cells with RNase eliminates the staining

9

suggesting that this protein associates with RNA in the nucleolus. Since ribosome production is

quite sensitive to heat shock, it is speculated that this protein is induced to protect the ribosomes

during heat shock (Nover et al., 1986).

The yeast HSP104, which is also localised in the yeast nuclei, has been shown to disaggregate

proteins in vitro and in vivo (Parsell et al., 1994). Unlike molecular chaperones such as the

HSP70 and HSP60, it does not prevent the aggregation of proteins (Parsell et al., 1993).

However, in co-ordination with HSP70 and HSP40, it has been shown to refold aggregated

proteins back to their native state (Glover and Lindquist, 1998).

1.3.2 HSP90 family of proteins

Members of the HSP90 family have been cloned and sequenced from evolutionarily diverse

organisms, including fruit flies, yeasts, chickens, mammals and trypanosomes. They are amongst

the most abundant proteins in the cytosol of eukaryotes and their abundance is further increased

by stress treatment (Georgopoulos and Welch, 1993). They are composed of a very highly

conserved 25 kDa N-terminal domain and a 50 kDa C-terminal domain separated by a stretch of

charged residues. They have been shown to be essential in two model eukaryotic species -

Saccharomyces cerevisiae and Drosophila melanogaster. In yeasts it is developmentally

regulated. It accumulates in cells as they transit into stationary phase or begin to sporulate. In the

fruit flies, this protein (HSP83) is developmentally induced during oogenesis (Zimmerman et al.,

1983). The protein supports several signal transducers and lies in the interface of several

developmental pathways. Recent developments have proposed that this protein acts as a

morphological capacitor in allowing mutations to be accumulated over a period of time and thus

10

help the organism to evolve during evolutionary change (Rutherford and Lindquist, 1998). In

humans there are 2 variants of this protein: one exists in the cytoplasm (87-92 kDa) and a larger

protein (94-108 kDa) exists in the endoplasmic reticulum (ER). The ER protein is induced by

glucose starvation and is quite often referred to as GRP94 (Sciandra and Subjeck, 1983).

1.3.3 HSP70 family of proteins

The proteins from this family have been relatively well studied. They are present in almost all

the species (except for some Archaea like Methanoccocus jannaschii). The HSP70 proteins bind

transiently to hydrophobic regions of extended polypeptides as these emerge in several processes:

either during synthesis from a tunnel in the ribosome, during transport from a lipid bilayer, or on

the surface of a folded protein as a result of conformational change that occurs during the normal

functioning of the cell as in the dissociation of the clathrin coats. Under normal growth

conditions, this binding serves a basic chaperone function by reducing the incorrect interaction

between transiently exposed hydrophobic surfaces. The bacterial protein DnaK, an HSP70

homologue, exhibits chaperone activity which is driven by an ATPase activity that regulates

cycles of polypeptide binding and release. The N-terminal domain ( 44 kDa) of this protein binds

ATP and is responsible for the ATPase activity of the protein, while its C-terminal domain ( 18

kDa) contains the peptide-binding site (Bukau and Horwich. 1998). The ATP-bound form of

DnaK has low affinity for the polypeptide while the ADP-bound form has a very high affinity for

the polypeptide (Schmid et al., 1994). Nucleotide hydrolysis is considered to be the rate-limiting

step in the ATPase cycle. The intrinsic ATPase activity of DnaK is very weak. There are other

proteins that are known to associate and influence the ATPase cycle. DnaJ and the GrpE families

of proteins, called co-chaperones, interact with DnaK and enhance the hydrolysis and nucleotide

11

release from the DnaK (Szabo et al., 1994) (see figurel.1A). This machinery is required in the

replication of the lambda bacteriophage (Georgopoulos et al., 1973).

Most eukaryotes have multiple HSP70 homologues. Yeast contains 8 well-characterised

HSP70s; six in the cytosol called ssa1-4 and ssb1-2, one in the mitochondrial matrix called ssc1p

and one in the ER lumen called the ssd1p or Kar2p. The ssa1-4 genes are induced during the heat

shock whereas the ssb1-2 genes are repressed. Mutations in the ssb genes render the

microorganism cold sensitive. Yeast cells deprived of the ssa proteins accumulate mitochondrial

and ER precursor proteins. In animal cells, the ER HSP70 is called BiP, since it binds to a wide

variety of nascent polypeptides imported in the ER from the cytosol. The binding of proteins is

usually transient unless there are mutations in the incoming protein, which would hinder its

folding/oligomerisation to the native state (Gething et al., 1986). A similar effect is seen when

the proteins entering the ER are not properly glycosylated.

1.3.4 HSP60 family of proteins

This class of chaperones are referred to as chaperonins (Hemmingsen, 1988). The folding

mechanisms of the chaperonins differ from the other chaperones (specifically HSP70). They have

a double torroidal structure and hence provide a central cavity in which the polypeptide molecule

folds. The folding of the substrate protein is initiated after the protein is released into the

cytosolic milieu. The mechanistic aspect of GroEL, the E. coli chaperonin, has been studied in

great details. The chaperonin works with the cofactor, GroES. GroEL is organised as a double

doughnut structure consisting of 14 subunits, 60 kDa each, which are isologously placed so that

the two rings of the doughnut together enclose a central cavity of about 15nm in length. Each

12

(a)

(b)

DnaJ u ATP

~~ \____ ) ~ ~~ [ ~# ..

P,; DnaJ ADP u

DnaK .. u)

" " ~ 7 ATP + GroES ~~ - ~# 7 ATP

\____ -15 s .. ~

[S \" 7 P, 7 ADP. GroES

Figure 1.1 Schematic representation of the folding mechanisms of

HSP70 (DnaK/DnaJ/GrpE) and HSP60 (GroEL/GroES). The

HSP70 family of proteins bind to unfolded polypeptides. Whenever

the unfolded polypeptide is released in free solution, folding is

initiated In contrast substrates for GroEL are partially structured

and folding occurs in the protected environment of the

GroEL/GroES cage.

N

#N §]

monomer consists of 3 domains. The large equatorial domain, which possesses the ATP binding

site, is linked to the other large, peptide binding apical domain via the smaller intermediate

domain. The polypeptide binds in a somewhat compact conformation to the hydrophobic patches

on the apical domain. GroES, a single heptameric ring of 10 kDa subunits, resembles an upturned

saucer. The GroES presents a largely unstructured mobile loop, which in the presence of

nucleotide interacts to form an asymmetric GroES7-GroEL14 complex. This creates a hydrophilic

cage often referred to as the Anfinsen's cage into which the polypeptide is released. The unfolded

polypeptide tries to fold to its native conformation by seeking free energy minima anew. The

GroES cap dissociates after every 20 seconds and the polypeptide is ejected into the cytosol. This

is facilitated by the binding of the ATP to the distal trans side of the GroEL (the cis side is the

side to which the GroES is bound with the unfolded polypeptide). Figure l.lB shows a schematic

representation of the entire GroEL cycle. Polypeptides may take more than one cycle to complete

their folding. Close homologues of GroEL and GroES are found in the mitochondria (HSP60 and

HSPIO) and chloroplasts (cpn60 andcpn10). The eukaryotic cytosol contains the chaperonin

TRiC or CCT (for TCP-1 ring complex and cytosolic chaperonin containing TCP-1 respectively).

It is a large hetero-oligomeric complex containing nine different subunits. The functioning of this

chaperonin is quite distinct from that of the GroEL-GroES system and is poorly understood. This

chaperonin does not have a co-chaperonin like GroES and is known to fold cytoskeletal proteins

such as tubulin (Gao et al., 1992).

1.3.5 Small heat shock proteins

The small Hsps encompass a group of proteins, which have molecular masses in the range of 12-

43 kDa. These proteins assemble into large heterogeneous multimers where the molecular mass

13

of the oligomer might increase upto 2000.kDa (Waters et al., 1996). They form a structurally

divergent protein family with members present in archaea, prokaryotes and eukaryotes. The small

Hsps are probably the least understood in terms of specific functions. They were considered to

be a product of an ancient viral DNA, which serves no function (Susek and Lindquist, 1989).

This appears to be very unlikely considering the fact that in plants and higher animals these Hsps

are the most highly induced of the stress proteins. Recent studies on these small heat shock

proteins have shown that they are capable of preventing the thermal or chemical-denaturant

induced aggregation of proteins in a way similar to the Hsp90. All these proteins have a

conserved C-terminal domain, which probably helps in the multimerisation of the protein, and

a highly variable N-terminal domain. In the human cells these small shock proteins have been

found to be associated with actin and intermediate filaments and hence are thought to regulate

the dynamics of these filaments. The family of small Hsps also includes a.-crystallin, which is

predominantly present in the eye lens.

1.3.5.1 Crystal structure of the sHSP

The crystal structure of the sHSP from Methanoccocus jannaschii, a hyperthermophilic archaeon

was recently solved (Kim et al., 1998). The protein is a hollow spherical complex composed of

24 subunits (aggregate size -400 kDa). Each subunit has a molecular weight of 16.5 kDa. The

subunits are related by an octahedral symmetry (figure 1.2) with a total of twelve two-fold, three

three-fold and three four-fold non-crystallographic symmetry axes, and one crystallographic

three-fold symmetry. The outer diameter of the sphere is 120 A and the inner diameter is 65 A.

The inside of the sphere is hollow. There are 8 triangular and 6 square windows on the surface

of the sphere. Each folding unit is composed of 9 ~-strands in two sheets, two short 310 helices

14

a

c

b

d

Figure 1.2 Subunit interaction of sHsp a) Topology of the secondary

structure of a MjHsp 16.5 dimer b) Ribbon diagram of the MjHsp

viewed along the non-crystallographic two-fold symmetry axis. c)

Three dimers related by a non-crystallographic three-fold symmetry d)

Overall structure of the sHsp, a space-filling model of the hollow

sphere viewed along the crystallographic three-fold axis.

and one short P-strand. One of the strands comes from the neighboring subunits. The amino

terminal residues are highly disordered. 41% of the solvent accessible surface in each monomer

is buried at inter subunit contacts. Of these, 48% of the contact surfaces contribute to dimer

formation, 42% to tetramer formation and only 10% to trimer formation. 66% of the dimer

contacts are due to non-polar interactions, the rest contributed by hydrogen bonds and ionic

-interactions. TheN-terminal residues are placed inside the sphere. Interestingly, 49% of the

solvent accessible surface in the interior of the sphere is composed of hydrophobic residues.

This structure of the small heat shock protein is quite different from the structure of the

chaperonin GroEL. The diameter of the central cavity 'in GroEL is about 47 A in diameter. The

length of the channel is 146 A and resembles a cylinder. If this channel were assumed to be a

smooth cylinder, the volume of the cylinder would be about 250,000 A3, which is about twice

in size of the cavity of the sHSP. If the partial specific volume of the protein is 1.73 A3 per unit

Mr then there would be enough space for a polypeptide of Mr 147 kDa provided it is fitted into

the entire channel with a perfect close packing. If the chaperonin is occupied by an unfolded

protein like the molten globule, which would occupy double the volume occupied by the native

protein, then the central channel would be able to accommodate a protein with a mass of about

50-60 kDa.

1.4 THE CHAPERONING NETWORK

Recently, there have been a considerable number of observations, which point out that there

exists a highly coordinated process involving the sequential and progressive interactions of

different chaperone systems with folding and unfolding intermediates, which are formed during

the life cycle of the cell. Similar evidences are found in bacterial systems where the bacterial

15

sHSP (IbpA) interacts with the HSP70 and HSP60 group of chaperones to recover the thermally

denatured proteins accumulating during thermal stress (Vienger et al., 1998).

1.4.1 Integration of the chaperone systems during protein biogenesis

The first indication that different chaperone systems might cooperate in folding processes came

from the studies on the mitochondrial import of proteins (Hohfeld and Hartl, 1994). As a

translocating polypeptide chain emerges into the mitochondrial matrix the mitochondrial HSP70

transiently interacts with the polypeptide. It provides· the driving force for translocation and

prevents the polypeptide from aggregating. The polypeptide is then transferred to the

mitochondrial HSP60 in the matrix, which folds the protein to its native conformation. Such

interactions have also been observed in the case of protein translocation into the chloroplast.

Mechanistic insight into the functional cooperation of these chaperone proteins was provided by

the in vitro reconstitution of the sequential interaction of a folding polypeptide with the bacterial

HSP70 and HSP60 homologues along with their cofactors (Langer et al., 1992). The question as

to what determines the interaction of a polypeptide substrate with the subset of molecular

chaperone system can have two answers. One possibility is the spatial availability of individual

chaperones. This depends on the subcellular localisation of chaperone and the levels of this

chaperone at any given time. The second major determinant would be the binding specificities

of individual chaperones. For example, HSP70 would only interact with extended polypeptides

while the HSP60 would interact with the so-called molten globule state of the protein. Thus,

structural features exposed by the folding polypeptide might influence the successive interaction

of different chaperone systems.

16

1.4.2 Folding the sterol binding receptor

One of the most complex systems where the chaperoning network is understood to a great extent

is the steroid aporeceptor complex (Stancato et al., 1996). This complex contains about 10

chaperones; each of these chaperones could individually be a part of a totally separate

chaperoning system, which come together and help the receptor to be in a high affinity binding

state. Some of the well-characterised chaperones involved are HSP90, HSP70, Hdj-1 (the human

DnaJ homologue), p60, p48 (HiP) and immunophilins~ The removal of any of these chaperones

from the heteromeric complex or disturbance of the interaction between components by adding

drugs such as geldanamycin disrupts the ability of the steroid receptor to bind to its ligand.

Individually, the chaperones such as Hsp90, p60 or p23 cannot refold the receptor or other

unrelated proteins like p-galactosidase. HSP90 can suppress the aggregation of the denatured

proteins by maintaining them in a soluble folding competent state. Hence these proteins form a

part of the chaperone network and modulate the ability of an oligomeric protein to stay in an

active conformation.

One way of understanding the role of a chaperone in the cell would be to look at the

conformational features that the chaperone recognises for proper interaction. The conformational

requirements of a number of chaperones have been well worked out.

1.4.3 Conformation of the interacting polypeptides

Rudiger and colleagues (1997) looked at 37 known substrates for the bacterial HSP70 (known

17

as DnaK) and found that the sequence that binds to DnaK probably has a consensus sequence

NRLLLTG. This sequence lies mostly in the unstructured regions in a growing or unfolded

polypeptide. Peptide binding to DnaK is mediated by hydrogen bonds between the chaperone

and the polypeptide backbone, as well as by hydrophobic contacts and possible salt bridges with

the side chains (Zhu et al., 1996). In general this binding is similar to that of antigens binding to

the MHC molecules.

Tr-NOE studies demonstrated that most of the peptides binding to GroEL had an alpha-helical

conformation (Landry et al., 1992). Proteins such as Citrate synthase, rhodanese and rubisco,

which are known targets for GroEL, have been shown to possess a solvent-exposed amphipathic

amino terminal alpha helix, which interacts with GroEL. Based on these studies it was assumed

that the side chain hydrophobicity and the innate capacity of the peptide to form a helix were the

only requirements a protein must have in order to bind GroEL. Schmidt and Buchner ( 1992), on

the contrary, showed that an all beta-sheet protein such as the Fab fragment of the

immunoglobulin could also interact with GroEL. The current understanding is that HSP60 binds

to compact folding intermediates, which expose hydrophobic surfaces (Hayer-Hartl et al., 1994).

These intermediates already have native-like secondary structure and lack proper tertiary

structure. Completely random coiled polypeptides such as reduced carboxymethylated alpha­

lactalbumin do not interact with the chaperonin.

18

1.5 JOINING THE CHAPERONE FAMI~Y; ALPHA CRYSTALLIN AS A

MOLECULAR CHAPERONE

1.5.1 The Eye Lens and a-Crystallin

The vertebrate eye lens is a transparent, highly refractive structure located between the pupillary

portion of the iris and vitreous. It is avascular and is devoid of lymphatics and nerves. The

epithelial cells in the posterior part of the eye lens elongate and terminally differentiate to form

the lens fibre cells. These fibre cells are devoid of cell organelles and contain high concentrations

of proteins called crystallins (300-500 mg/ml). As the lens grows, newly formed fibre cells are

added to the pre-existing fibres. The newly formed fibres occupy the cortical region of the eye

lens (peripheral region) while the older cells are pushed towards the nucleus. Thus, at any age

the internalised concentric layers of the lens reflect the processes of the fibre cell differentiation,

maturation and ageing of the lens cells. Cataract is a disease condition wherein the lens loses its

transparency.

There are three major crystallins namely, alpha, beta and gamma. Besides these, there are a

number of taxon-specific crystallins such as delta-, rho-, epsilon- and zeta-crystallins, which

possess enzymatic activities besides being a part of this refractile tissue. The crystallins are

packed in an orderly and spatial fashion in the terminally differentiated lens fibre. The spatial

arrangement in the lens is set up through the differential over-expression of the a-, ~-. y­

crystallins in a location-specific manner during the lens development. This sets up a gradient of

refractive index varying from 1.37 in the periphery (cortex), where the concentration is 150-200

mg/ml, to 1.46 in the core (nucleus), where the protein concentration is about 500 mg/ml. This

19

spatial ordering of the crystallins is probably requireg for minimising the spherical aberrations

and light scattering (Guptasarma, 1993).

a-Crystallin is one of the major eye lens proteins which is coded by the genes aA and aB. The

most striking feature of this protein is the size of the multimer which is around 800 kDa. The

gene products of aA and aB are 20 kDa (173 and 175 amino acid residues respectively) and

share 54% amino acid identity (Vander Ouderaa et al., 1974), suggesting that these two genes

arose from a common ancestor. aA and aB crystallins are present as a heteroaggregate (a-

• crystallin) in the lens in a ratio of 3:1 respectively. This ratio varies with species and with age.

a-Crystallin is a predominantly beta sheeted protein which forms spherical aggregates with an

average diameter of about 140-150 A. The crystal structure of this molecule is not known and

hence the quaternary structural arrangements and packaging details remain a matter of

controversy. Based on the crystal structure of the sHSP one can speculate that this molecule

would have a structure quite similar to the sHSP. The major differences would be in the size of

the internal cavity and the number of N-terminal residues occupying the cavity.

lngolia and Craig ( 1982) were the first to show that the sHSP of Drosophila shares conspicuous

sequence similarities with a-crystallin, reflecting a common evolutionary ancestry. Soon it was

found that a large number of sHSPs from very diverse species of living organisms shared

sequence homology with a-crystallin. The humans have 5 paralogous proteins: aA, aB, HSP20,

HSP27 and the more recently discovered HSPL27 which have all originated from a common

ancestral gene by gene duplication. Quite often, in tissues expressing more than one type ofHSP,

these proteins are isolated as heteromeric complexes. Proteins can be considered as evolutionarily

20

related when sequence similarities can be identified over long and contiguous stretches of

residues. Members of the a-crystallin family are characterised by the presence of a homologous

sequence of 80-100 amino acid residues, the a-crystallin domain, in their C-terminal region. An

uninterrupted block of sequence similarity exists between the a-crystallins and the HSP27 from

positions 150 to 235 of the HSP 27. Many of the sHSP genes from prokaryotes, Streptomyces

and Leuconostoc to higher eukaryotes are constitutively expressed at low levels and become

upregulated under conditions of stress probably conferring stress-tolerance. Some of the sHSPs,

as in Clostridium, cannot be induced by exogenous stresses. They are developmentally regulated

• and are assumed to be involved in coping up with the wear and tear during normal growth. Thus,

the function of the super family of a-crystallin seems to be to help the cell to survive under the

- stress of life, whether transient or constitutive.

1.5.2 Expression of a8 crystallin in non-lenticular tissues

Bhat and Nagineni (1989) showed that the expression of a-crystallins was not restricted to the

eye lens. It can be found in the kidney, brain, heart and striated muscle (slow-twitch muscles).

The levels of a-crystallin increase in these tissues under pathological conditions such as

ischemia, brain tumour or during oncogenic transformation of cells in culture. Thus, the myth

that a-crystallins were mere structural proteins was eliminated. It was further shown that a.B-

crystallin is induced in cell lines when the cells are subjected to heat shock (Aoyama et al., 1993)

or a hypertonic shock (Dasgupta et al., 1992). a.B-crystallin exists in low levels in tissues even

under non-stressed conditions. About 3-5% of the total soluble protein in the cardiac tissue is a.B-

. crystallin. Immunocytochemical localisation of a.B-crystallin has revealed that this protein is

21

found only in the central portion of the I bands. aB-crystallin appears as a major contaminant in

the cardiac desmin preparations (Bennardini et al., 1992). Intermediate filaments (IFs) constitute

a major part of the cytoskeletal components in the eukaryotic cytoplasm and nuclear lamina. IFs

are radially distributed from the nuclear membrane towards the cell membrane. The IFs collapse

towards the nucleus and form a large juxta nuclear cap when subjected to heat shock or treated

with certain drugs. The levels of expression aB-crystallin under these conditions is upregulated

and it interacts directly with the IFs probably helping the filaments to reorganise after the stress

is removed (Djabali et al., 1997).

1.5.3 Chaperone-like properties of a-crystallin

The first direct evidence that a-crystallin could function as a molecular chaperone came from

Horwitz's laboratory (Horwitz, 1992). He showed that a-crystallin was able to prevent thermal

and chemical denaturant-induced aggregation of both lenticular and non-lenticular proteins in

vitro. The choice of a-crystallin as one of the major lens crystallin seems to be an example of

gene sharing, whereby one gene encodes a protein with two functions (Piatigorsky and Wistow,

1989). The primary function of this protein in the lens could be to form a part of the lens

contributing to its stability and refractile properties and the other function could be to act as a

chaperone and prevent the aggregation of the other lens proteins. In the other non-lenticular

tissues, where a-crystallin is expressed, the protein might serve as a molecular chaperone and

help cells overcome stress.

22

1.5.3.1 Temperature-dependent chaperoning properties of a-crystallin

The compact architecture of a protein becomes destabilised during thermal aggregation. In order

to rule out structural alterations in a-crystallin, our laboratory investigated non-thermal modes

of aggregation. a-Crystallin is capable of preventing aggregation of proteins in a temperature-

dependent manner. The chaperoning activity of a-crystallin was found to be poor at low

temperatures (below room temperature) but increased significantly above 30°C. This study

indicated that a-crystallin might undergo a structural transition at this temperature and start

-. exposing more and more hydrophobic surfaces as the temperature increased. Based on these

results it was hypothesised that a-crystallin undergoes a structural perturbation, probably a

rearrangement in the quaternary structure, which enables the molecule to protect the proteins

from denaturing by providing appropriately placed hydrophobic surfaces (Raman and Rao, 1997;

Rao et al., 1998).

1.5.3.2 Structural perturbation of a-crystallin

y-Crystallin aggregates upon exposure to UV-light. a-Crystallin is able to prevent the aggregation

of y-crystallin in a temperature-dependent manner. It offers no protection at temperatures below

30°C and completely protects the aggregating protein at about 55°C. This non-monotonic

behaviour of a-crystallin towards protecting the target protein suggests a structural transition

around this point. Several studies on the chaperoning ability have suggested that a-crystallin can

be perturbed with changing temperature, ionic strength and pH (Siezen et al., 1980). Differential

scanning calorimetric studies has shown two broad endothermic transitions, one at around 40°C

23

Table 1.1 Free energies of transfer of pyrene from buffer to

a-crystallin

S.No Temperature, oK .LGtr (cal.moi-1)

1. 291 -1055

2. 312 -1427

328 -1905 3.

4. 336 -2070

All measurements were made with a 0.3 mg/ml solution of a­

crystallin in 10 mM phosphate buffer of pH 7.2 containing 100

mM NaCl. Concentrations of pyrene were measured using

absorbance values at 338 nm . .LGtr were calculated from the

equation, .LGtr = -RT In (CjCb); were Cb and Ca are solubilities

in buffer and in the a-crystallin solution. (adapted from Ph.D

thesis Raman, 1997)

and the other at around 60°C - the first transition appears to depend upon the concentration of

the protein. Perturbation studies using 3 M urea show that the protective ability of a-crystallin

increases at room temperature in the presence of the denaturant. Our laboratory investigated the

accessibility of the hydrophobic surface of a-crystallin by solubilising the hydrophobic

fluorophore, pyrene (Raman and Rao, 1994). The free energy of transfer ~Gtris presented in a

tabular form (Table 1.1 ). The intensity ratio of the vibronic emission peak 3 to the vibronic

emission peak 1 of the fluorescence spectrum of pyrene reports the microenvironment around the

probe. In water, the ratio is about 0.5 and in hydrophobic conditions it increases to. 1.0. It is

evident from the figure (figure 1.3) that a-crystallin offers considerable surface hydrophobicity.

The ratio increases to about 0.8 at around 40°C. Similar results demonstrating the structural

transition in a-crystallin were also seen while investigating the chaperone-like activity of a­

crystallin towards protecting DTT-induced aggregation of insulin (Raman et al., 1995) and

refolding-induced aggregation of ~-crystallin (Raman et al., 1995). Thus, the accessibility of the

hydrophobic surface of a-crystallin, as reflected in the enhanced solubility of pyrene, increases

with temperature and parallels the temperature-dependent protective ability.

1.5.3.3 Chaperoning by aA- and aB-crystallin

Individual chaperoning properties of aA- and aB-crystallin are quite different from the

chaperoning property of a-crystallin. Studies on this aspect might shed light on the differential

roles of this protein in the lens and in other non-lenticular tissues. Datta and Rao (1999) have

shown that the exposed hydrophobic surfaces of aA-crystallin, aB-crystallin and a-crystallin are

quite different at room temperature. aB-crystallin exposes maximal hydrophobic surfaces and

24

I I

0.10

QIO

~ 0.06t­Q ----­r•··

• . / . 0 •

·-· I 0.86 1.

5 • ,_.___.., ;· • 0'--~......_ _ _.__...........,.-'--o-o~y

350 370 ;)90 41 0 430

WAVELENGTH (nm) ·-· . · .. . / ./

.~ 0. 78 '-------'-----'-----'-...................... ----Ji.--'--__.__ .................

10 30 50 70

Temperature (°C)

Figure 1.3 Accessibility of hydrophobic surfaces of a-crystallin

with temperature. (A) Solubility of pyrene in a-crystallin with

temperature. (B) The ratio of the vibronic emission peak 3 to peak

1 of fluorescence spectrum of pyrene solubilized in a-crystallin

with temperature. Inset shows the fluorescence spectra of pyrene

in water and in methanol. (Adapted from Ph.D. thesis Raman,

1997)

is capable of preventing aggregation of proteins at ,x:oom temperature whereas both a.A-crystallin

and a-crystallin protect sparsely at this temperature. The structural stability of aB-crystallin is

less as compared to the other two forms of crystallin and it loses almost all its tertiary structure

by about 50°C. The ratio of aB-crystallin to a.A-crystallin in the lens is 1:3 and probably reflects

the reason why the protein is so stable in the eye lens. In the non-lenticular tissues where stability

may not be of prime importance, and the ability of protecting aggregating proteins might be of

primary importance, only aB-crystallin homoaggregates are expressed.

1.5.3.4 a-Crystallin and disease

As mentioned earlier aB-crystallin interacts with intermediate filaments and probably helps in

maintaining the integrity of these filaments. Recent studies on desrnin related myopathies (DRM)

have shown that aB-crystallin coaggregates with desrnin filaments to form aggregates. Patients

suffering from DRM have mutant forms of aB-crystallin or desmin, which leads to skeletal

muscle weakness, and are prone to heart failure. One of these mutants (R120G) has been

characterised (Bova et al., 1999). The aberrant protein forms bigger and more disperse multimers

than the native molecule and has reduced chaperone-like activity. These patients also suffer from

lens opacification (cataract) where the concentrations of these proteins are probably the highest.

A corresponding mutation in a.A-crystallin (R116C) causes congenital cataract (Litt et al., 1998;

Kumar et al., 1999). This form of a.A-crystallin has been shown to form intermolecular

disulphide bonds (Shroff et al., 2000). Besides DRM, aB-crystallin co-aggregates in the case of

Alexander's disease, which is characterised by the presence of Rosenthals fibres, and alcoholic

hepatitis, which is characterised by the presence of Mallory bodies. Their expression has been

25

observed in scrapie-infected hamster brain .cells (Duguid et al., 1988), in Nlli3T3 mouse

fibroblasts expressing Ha-ras and v-mos oncogenes (Klemenz et al., 1991), and in fibroblasts

from patients with Werner syndrome (Murano et al., 1991).

1.6 SCOPE OF THE PRESENT STUDY

a-Crystallin constitutes a major part of the eye lens and is expressed in elevated levels in non­

lenticular tissues under stress. It is known to bind to aggregating and damaged proteins and helps

tissues recover from acute stress caused during conaitions like ischemia, renal failure and

neurodegeneration. The molecular details of the chaperoning mechanism are not understood.

Earlier studies from our laboratory have suggested that a structural perturbation above 30 oc

enhances the ability of a-crystallin to prevent thermal and non-thermal aggregation of proteins.

This perturbation probably involves a reorganisation of the subunits, which makes available

appropriately placed hydrophobic ,surfaces to which the denaturing protein can bind.

Molecular chaperones in the cell fold nascent polypeptides in a highly co-ordinated sequential

manner. During biogenesis each chaperone recognises a specific partially folded intermediate and

through the progressive interactions between chaperones, a completely folded functionally active

protein is made. One way of understanding the role of a chaperone in the cell is to decipher the

conformational features that the chaperone recognises for proper interaction. The conformational

specificity for the Hsp70 and Hsp60 class of chaperones is well worked out. Hsp70 binds to

extended polypeptides showing a consensus sequence NRLLLTG. The chaperonins bind to a

more structured intermediate showing characteristics of a molten globule. The conformational

requirements of a-crystallin and the other small heat shock proteins are not known.

26

The present thesis aims at understanding the nature of interaction between an aggregating protein

(target protein) and a-crystallin. Characterisation of the conformational requirements involves

study of the interaction with proteins whose intermediates are well characterised. We have

investigated the interaction of a-crystallin with carbonic anhydrase, bovine a-lactalbumin and

citrate synthase.

Equilibrium state intermediates of carbonic anhydrase have been well characterised (Ptitsyn,

1995). Rao and Zigler (1993) investigated the thermal aggregation of carbonic anhydrase and

. showed formation of a stable complex between the chaperone and the enzyme. We have

investigated the intermediate of carbonic anhydrase, which is formed during thermal denaturation

and its interaction with a-crystallin. We have characterised this intermediate and compared the

conformation of a-crystallin-bound enzyme with the equilibrium state intermediates. These

studies indicated that a-crystallin probably binds to a molten globule-like intermediate. We have

also investigated the role of the metal ion zinc, which occupies the catalytic site in modulating

the propensity of the enzyme to go into a molten globule state. We find that the bovine and the

human isoforms of carbonic anhydrase show differences in thermal- and denaturant-induced

aggregation despite sharing high sequence homology. This difference may be attributed to the

tightness with which zinc binds the enzyme. Consequently the amount of a-crystallin required

to prevent these two isoforms are different.

a-Lactalbumin can be unfolded to non-native states under different experimental conditions to

yield distinct intermediates. These intermediates have varying residual secondary and tertiary

structural elements (Kuwajima, 1996). We have investigated the interaction of three distinct non-

27

native intermediates with a-crystallin. We have also investigated the role of charges in

modulating the chaperoning ability of a-crystallin. These studies have allowed us to establish the

conformational requirements for a-crystallin.

Small heat shock proteins and Hsp90 are known to prevent thermal denaturation of proteins and

help reactivate the enzyme (Jakob et al., 1993; Jakob et al., 1995). The lens a-crystallin is a

heteromultimer made up of 2 subunits aA- and aB-crystallin. These subunits are capable of

forming homopolymers, which are distinct in terms of stability and chaperoning ability in vitro

. (Datta and Rao, 2000). The thermoprotective abilities of these two homopolymers have not been

investigated and the molecular mechanisms involved in conferring the protective effect is poorly

understood. We have investigated the thermoprotective abilities of these two subunits

individually and the ability to reactivate early unfolding intermediates of the dimeric enzyme,

citrate synthase. Our investigations indicate that aB-crystallin is capable offering

thermoprotection better than aA-crystallin.

The ability of a-crystallin to prevent thermal and non-thermal aggregation and its presence in

non-lenticular tissues and overexpression in response to stress clearly shows that it functions as

a molecular chaperone in vivo. However, not much is known as to how this protein performs the

chaperoning function in vivo as there is no evidence either in vitro or in vivo to show that this

protein is capable of refolding the partially denatured molecule which is complexed to it. These

studies would enable us understand the mechanistic aspects of chaperone functions in general and

the role of a-crystallin in maintaining lens transparency. These studies would further help us

develop an in vivo assay system to investigate chaperoning mechanism of a-crystallin. Such a

28

system would help us screen rapidly and characterise mutants of a-crystallin, which are known

to cause congenital cataract and desmin related myopathy.

29