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PubMed1995

The roles of molecular chaperones in vivo.

Table 1 summarizes the families of chaperones mentioned in this review, and lists their proposed functions. Many of these proteins are named in the accompanying review of Burston and Clarke. Molecular chaperones are proteins which interact with other proteins and help them to reach their final, active conformation. They appear to do this by binding them in an unfolded or partially folded state and subsequently releasing them in an altered form. This property may endow them with several essential or important roles in addition to helping newly synthesized proteins to fold correctly, such as repairing damaged proteins and assisting proteins in membrane translocation. To confirm that a given protein has molecular chaperone activity in vivo, it is necessary to show that interactions between the chaperone and other proteins do occur in the cell, and that loss of the molecular chaperone leads to the accumulation of inactive or precursor protein. The hsp70 protein family are highly conserved and ubiquitous. Genetic studies confirm that their depletion leads to the accumulation of inactive precursor or other proteins, and immunochemical studies show they associate with nascent polypeptides. They are implicated not only in protein folding, but also in protein transport across membranes and reactivation of heat-damaged proteins. The hsp60 proteins are also ubiquitous and very similar in sequence. Those found in bacteria and organelles, such as mitochondria (the GroEL family), are essential at all temperatures, and particularly after heat shock. Their loss or depletion leads to the formation of protein aggregates and eventual cell death. A co-chaperone protein (GroES) is required for their function. Cytosolic homologues (the TCP1 family) are also essential, though not heat-shock induced; they are believed to have a chaperone role in tubulin assembly and their actual role in the cell may be much broader. Many other proteins may have a chaperone function in vivo. Such a function may be specific to a particular substrate (such as the PapD protein in E. coli); others may be more general (such as hsp90 and SecB). Evidence is still needed to demonstrate whether all those proteins which show chaperone behaviour in vitro actually have such a role in vivo. It seems likely that different classes of chaperone may overlap in their specificity, and it is certain that the various proteins classed as molecular chaperones fulfil a wide variety of roles in the cell.

Chaperonin 10↗

PubMed1998

How chaperones fold proteins.

Chaperones are a functionally related group of proteins assisting protein folding in the cell under physiological and stress conditions. They share the ability to recognize and bind nonnative proteins thus preventing unspecific aggregation. The underlying functional principles of the different chaperone classes are beginning to be understood. A landmark feature of molecular chaperones is the involvement of energy-dependent reactions in the folding process. Nucleotide binding to ATP-dependent chaperones (e.g. GroEL, Hsp70, Hsp90) leads to sometimes large conformational changes in the chaperone which allow to shift between high- and low-affinity states for substrate proteins. Interestingly, the ATPase activity which is the key determinant for functional cycles is tightly regulated by a set of co-chaperones. While for ATP-dependent chaperones binding sites for nucleotide and protein are found in one protein, in the case of ATP-independent chaperones (e. g. sHsps, SecB) the energy-dependent step is performed by another chaperone (Hsp70, SecA). Therefore, the ATP-independent chaperones can be regarded as efficient 'holding' components. Cooperation of different chaperone machineries creates a synergistic network of folding helpers in the cell, which allows to maintain protein homeostasis under conditions nonpermissive for spontaneous folding.

Models, Chemical↗

PubMed1993

Prions and molecular chaperones.

Molecular chaperones are proteins involved in the folding of other proteins. Among these chaperones, some are involved in their own folding (auto-chaperones). A question arises: what is the mechanism of the chaperone folding catalysis? A model for protein folding that uses the thermodynamics of irreversible processes and statistical mechanics to describe the phenomenon is proposed; the analysis presents a clear link between these two aspects. A consequence of this model is the possible existence of misfolded proteins. This point is discussed and some experimental results arguing in this direction detailed. This thermo-kinetic model is applied to protein folding driven by a molecular chaperone. Analysis of folding shows that a misfolded chaperone can induce misfolding in protein and, in the case of autofolding (auto-chaperone), may lead to new misfolded chaperones. The consequences are explored by computer simulations. They show that such an auto-chaperone could behave as a new kind of informative molecule and replicate a misfolded structure by a process similar to infection. A quantitative model, displaying the epidemiologic characters of prion infections, is derived from this hypothesis. This hypothesis satisfactorily explains the three manifestations (infectious, genetic and sporadic) that are the characteristic features of all prion diseases. Are prions really molecular chaperones required for their own assembly? Analysis of the structure of prions revealed some features shared by true molecular chaperones. This analysis suggests the positions of the mutations likely to lead to the characteristic early onset of encephalopathy. They are in good agreement with experimental results.

Amino Acid Sequence↗

PubMed2006

Folding on the chaperone: yield enhancement through loose binding.

A variety of small cageless chaperones have been discovered that can assist protein folding without the consumption of ATP. These include mini-chaperones (catalytically active fragments of larger chaperones), as well as small proteins such as alpha-casein and detergents acting as "artificial chaperones." These chaperones all possess exposed hydrophobic patches on their surface that act as recognition sites for misfolded proteins. They lack the complexity of chaperonins (that encapsulate proteins in their inner rings) and their study can offer insight into the minimal requirements for chaperone function. We use molecular dynamics simulations to investigate how a cageless chaperone, modeled as a sphere of tunable hydrophobicity, can assist folding of a substrate protein. We find that under steady-state (non-stress) conditions, cageless chaperones that bind to a single substrate protein increase folding yields by reducing the time the substrate spends in an aggregation-prone state in a dual manner: (a) by competing for aggregation-prone hydrophobic sites on the surface of a protein, hence reducing the time the protein spends unprotected in the bulk and (b) by accelerating folding rates of the protein. In both cases, the chaperone must bind to and hold the protein loosely enough to allow the protein to change its conformation and fold while bound. Loose binding may enable small cageless chaperones to help proteins fold and avoid aggregation under steady-state conditions, even at low concentrations, without the consumption of ATP.

Computer Simulation↗

PubMed2003

Enhancement of chaperone function of alpha-crystallin by methylglyoxal modification.

The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

Alcohol Dehydrogenase↗

PubMed2002

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

Amino Acid Sequence↗

PubMed2004

Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export.

Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells. The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus. We have identified a stalled intermediate export complex in the flagellar type III export pathway of Salmonella typhimurium by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium. These chaperone variants bound their specific export substrates strongly and severely reduced their export. They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway. Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus. Membrane localization persisted in fliOPQR, flhB, flhA, fliJ, and fliH null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI. After expression of the variant chaperones in Salmonella, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated. Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate. Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.

Adenosine Triphosphatases↗

PubMed2006

A genomewide analysis of genes for the heat shock protein 70 chaperone system in the ascidian Ciona intestinalis.

Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.

Animals↗

PubMed1999

Medical and osteopathic boards' positions on chaperones during gynecologic examinations.

OBJECTIVE: The objective of this study was to determine whether United States medical or osteopathic boards have opinions, position statements, or policies on chaperone use, and whether any state laws regulate chaperone use. METHODS: United States Medical and Osteopathic Boards were surveyed by mail to determine whether policies, opinions, positions, or laws exist regarding use of chaperones during gynecologic examinations. We sent the survey to executives at 67 state boards, identified by a list from The Federation of State Medical Boards. Our main outcome measure was positive response to the survey questions. RESULTS: Of 67 targeted sites, 61 responded (91%). Fourteen sites (23%) reported having informal or unpublished opinions recommending chaperones. Eleven sites (18%) reported having positions related to chaperones that have been published for their physicians. Four sites (6.5%) reported having policies specifically related to chaperone use. Thirty-two sites (52.5%) reported that they do not have opinions, positions, or policies related to chaperone use. No site reported state laws governing chaperone use. CONCLUSION: Response to our survey showed no concensus among state medical boards on the use of chaperones, leaving doctors and patients to decide for themselves whether they want or need chaperones present during gynecologic examinations.

Female↗

PubMed2005

Improvement of the production of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system.

A stable Tn-5B1-4 insect cell line co-expressing the recombinant GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 (GFPuv-beta3GnT2) protein fused to a melittin signal sequence with a lectin-like molecular chaperone, human calnexin (hCNX) or human calreticulin (hCRT), was constructed. The expression of either of these molecular chaperones is under the control of a weak promoter, OpMNPV IE2, while that of GFPuv-beta3GnT2 is under the control of Bombyx mori actin promoter. This co-expression system was compared between two different insect cell-baculovirus expression systems: (1) co-infection of the recombinant baculovirus containing a molecular chaperone (AcNPV-hCNX or -hCRT) with a recombinant baculovirus containing GFPuv-beta3GnT2 fused with the melittin signal sequence (AcNPV-me-GGT); (2) infection of AcNPV-me-GGT to a stably expressing cell line for either hCNX or hCRT. In the co-infection system, the intracellular GFPuv-beta3GnT2 expression level was low because of the improved secretion level ratio of the fusion protein, due to the chaperone expression. In the case of infection to the stably expressing cell line for a chaperone, the extracellular GFPuv-beta3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process beta3GnT2. On the other hand, the co-expression system produced an extracellular beta3GnT activity of 22-23 mU/mL, which was approximately 3.5- and 11-fold higher than those of the stable expression of the fusion gene without the chaperone and the conventional BES with the addition of protease, respectively. The secretion level ratio of the fusion protein of this system increased to 82%, which was approximately 1.5-fold that of any other expression system investigated thus far. These results indicate that the ratio of the expression level of the target gene to that of the chaperone gene may be an important factor in maximizing the production of a target protein. The molecular-chaperone-assisted expression system using a stably transformed insect cell line offers promising prospects for the efficient production of recombinant secretory proteins in insect cells.

Animals↗

PubMed2001

On the equilibrium between monomeric alpha-lactalbumin and the chaperoning complex of alpha-crystallin.

In chaperoning dithiothreitol-denatured alpha-lactabumin, alpha-crystallin forms a chaperoning complex. In order to study the kinetics of such chaperoning it needs to be established whether the formation of the chaperoning complex is a reversible or irreversible process. The chaperoning reaction was studied by dynamic light scattering as a function of concentration and weight ratio of alpha-lactalbumin/alpha-crystallin. HPLC and subsequent SDS-PAGE gel electrophoresis experiments established that the chaperoning complex formed contains both alpha-crystallin and alpha-lactalbumin. Upon rechromatographing the chaperoning complex, the presence of monomeric alpha-lactalbumin has been demonstrated in addition to the chaperoning complex itself. This and equilibrium dialysis experiments demonstrated conclusively the existence of an equilibrium between monomeric partially denatured alpha-lactalbumin and the chaperoning complex made of alpha-lactalbumin and alpha-crystallin.

Animals↗

PubMed1997

The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system.

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK ATP; (2) DnaJ-triggered conversion of peptide x DnaK x ATP (T state) to slowly-acting, high-affinity peptide x DnaK x ADP x P(i) (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T --> R and R --> T conversion were 0.04 s(-1) and 1.0 s, respectively. The clearly rate-limiting T --> R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T --> R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.

Adenosine Triphosphatases↗

PubMed2003

Aging and molecular chaperones.

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer's disease, Parkinson's disease, Huntington's disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Aging↗

PubMed1998

Mechanistic comparison of artificial-chaperone-assisted and unassisted refolding of urea-denatured carbonic anhydrase B.

BACKGROUND: We have previously described a method for the refolding of chemically denatured proteins in which small molecules ('artificial chaperones', a detergent and cyclodextrin) assist renaturation. In a previous analysis of lysozyme refolding from the GdmCl-denatured, DTT-reduced state, we found that enzymatic activity is regained at indistinguishable rates for unassisted (absence of additives) and artificial-chaperone-assisted refolding. While unassisted and artificial-chaperone-assisted refolding rates could also be directly compared for GdmCl-denatured bovine carbonic anhydrase B (CAB), only cationic detergents could be used as assistants. We therefore set out to determine whether artificial chaperones could assist the refolding of urea-denatured CAB, whether the charge and structure of the detergent used affects refolding assistance, and, if so, whether the assistance is mechanistically similar to that observed for GdmCl-denatured CAB. RESULTS: Our results indicate that CAB can be refolded from the urea-denatured state via the artificial chaperone process, using both anionic and cationic detergents. There is a distinctive product-determining step early in the artificial-chaperone-assisted refolding mechanism, but the rate-determining steps of the unassisted and artificial-chaperone-assisted processes are indistinguishable. CONCLUSIONS: Because the rate-determining steps of unassisted and artificial-chaperone-assisted refolding are indistinguishable, we conclude that the rate-determining step of CAB refolding is unaffected by the use of artificial chaperones. Our observations also suggest that denatured CAB undergoes a slow partial folding in concentrated urea solution.

Animals↗

PubMed2006

Mini-alphaB-crystallin: a functional element of alphaB-crystallin with chaperone-like activity.

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.

Amino Acid Sequence↗

PubMed1996

Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.

Renaturation of thermally denatured firefly luciferase in rabbit reticulocyte lysate (RRL) requires hsp90, hsc70, and other as yet unidentified RRL components [Schumacher, R.J., et al. (1994) J. Biol. Chem. 269, 9493-9499]. Benzoquinonoid ansamycins (BAs) have recently been shown to specifically bind hsp90 and inhibit its function. In this report, we present data that indicate BAs are specific inhibitors of hsp90 function. The effects of the BA geldanamycin (GA) on the kinetics of the luciferase renaturation in RRL were examined to gain insight into the mechanism by which GA inhibits the function of the hsp90 chaperone machinery. Chaperone-mediated renaturation of luciferase obeyed Michaelis-Menten kinetics. The GA inhibited luciferase renaturation uncompetitively with respect to ATP concentration and noncompetitively with respect to luciferase concentration, indicating that GA binds after the binding of ATP and that it binds to both the hsp90 chaperone machine/ATP complex and the hsp90 chaperone machine/ATP/luciferase complex. GA markedly decreased the Kapp of the hsp90 chaperone machine for ATP, suggesting that GA increases the binding affinity of the hsp90 chaperone machinery for ATP or it slows the rate of ATP hydrolysis. Consistent with the notion that GA specifically binds hsp90 and inhibits its function, addition of hsp90, but not hsc70, p60, or p23, reversed GA-induced inhibition of luciferase renaturation in RRL. Hsp90, hsc70, and the hsp cohorts p60, p48, and p23 were coimmunoprecipitated with luciferase from RRL. GA increased the steady-state levels of luciferase associated with hsp90/hsp70 chaperone machine complexes that contain p60 and blocked the association of the hsp90 cohort p23 with chaperone-bound luciferase. The data suggest that the function of the hsp90 chaperone machinery is not specific to its previously described interaction with steroid hormone receptors, and that it carries out some more generalized function in vivo.

Animals↗

PubMed2005

Casein proteins as molecular chaperones.

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

Anilino Naphthalenesulfonates↗

PubMed2000

Targeting and insertion of C-terminally anchored proteins to the mitochondrial outer membrane is specific and saturable but does not strictly require ATP or molecular chaperones.

A distinct class of proteins contain a C-terminal membrane anchor and a cytoplasmic functional domain. A subset of these proteins is targeted to the mitochondrial outer membrane. Here, to probe for the involvement of a saturable targeting mechanism for this class of proteins, and to elucidate the roles of chaperone proteins and ATP, we have utilized an in vitro targeting system consisting of in vitro-synthesized proteins and isolated mitochondria. To establish the specificity of targeting we have used a closely related protein pair. VAMP-1A and VAMP-1B are splice variants of the vesicle-associated membrane protein/synaptobrevin-1 (VAMP-1) gene. In intact cells VAMP-1B is targeted to mitochondria whereas VAMP-1A is targeted to membranes of the secretory pathway, yet these isoforms differ by only five amino acids at the extreme C-terminus. Here we demonstrate that, in vitro, VAMP-1B is imported into both intact mitochondria and mitochondrial outer-membrane vesicles with a 15-fold greater efficiency than VAMP-1A. We generated and purified bacterially expressed fusion proteins consisting of the C-terminal two-thirds of VAMP-1A or -1B proteins fused to glutathione S-transferase (GST). Using these fusion proteins we demonstrate that protein targeting and insertion is saturable and specific for the VAMP-1B membrane anchor. To elucidate the role of cytosolic chaperones on VAMP-1B targeting, we also used the purified, Escherichia coli-derived fusion proteins. (33)P-Labelled GST-VAMP-1B(61-116), but not GST-VAMP-1A(61-118), was efficiently targeted to mitochondria in a chaperone-free system. Thus the information required for targeting is contained within the targeted protein itself and not the chaperone or a chaperone-protein complex, although chaperones may be required to maintain a transport-competent conformation. Moreover, ATP was required for transport only in the presence of cytosolic chaperone proteins. Therefore the ATP requirement of transport appears to reflect the participation of chaperones and not any other ATP-dependent step. These data demonstrate that targeting of C-terminally anchored proteins to mitochondria is sequence specific and mediated by a saturable mechanism. Neither ATP nor chaperone proteins are strictly required for either specific targeting or membrane insertion.

Adenosine Triphosphate↗