Structural characterization of the partially folded intermediates of an immunoglobulin light chain leading to amyloid fibrillation and amorphous aggregation

Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.     

A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP. By fitting our circular dichroism data to a model in which TFE-water mixtures are assumed to be ideal solutions, we show that increasing entropic costs of protein solvation in TFE-water mixtures may both cause the population of the intermediate state and increase aggregate production. Tertiary structure and electrostatic repulsion also impede aggregation. We conclude that initiation of EGFP aggregation in TFE likely involves overcoming of multiple protective factors, rather than stabilization of aggregation-prone structural elements.     

Dissecting the hydrogen exchange properties of insulin under amyloid fibril forming conditions: a site-specific investigation by mass spectrometry

We have examined the hydrogen exchange properties of bovine insulin under solution conditions that cause it to aggregate and eventually form amyloid fibrils. The results have been obtained at the residue-specific level using peptic digestion and mass spectrometry. A total of 19 peptides were assigned to regions of the protein and their exchange properties monitored for a period of 24 hours. The results of the peptic digestion show that residues A13 to A21 and B11 to B30 are more susceptible to proteolysis than the N-terminal regions of the protein. A total of 15 slowly exchanging amides were observed for insulin under these solution conditions. Location of the protected amides was carried out using a peptic-digestion protocol at low pH. Chromatographic separation was not required. This enabled a direct comparison of the peptides within the same mass spectrum. From kinetic analysis of the rates slow exchange has been located to 4(+/-1) backbone amides in the A13-A19 helix and 6(+/-1) in the B chain helix. The remaining 5(+/-1) are assigned to helix A2-A8. Taken together the results from digestion and hydrogen exchange show that at low pH and relatively high concentrations the C termini of both chains are susceptible to proteolysis but that the solution structure contains the native state helices. More generally the results demonstrate that mass spectrometry can be applied to study site-specific hydrogen exchange properties of proteins even under conditions where they are known to be partially folded and aggregate extensively in solution.     

Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases

Mutations in human copper zinc superoxide dismutase (hSOD) that are associated with amyotrophic lateral sclerosis (ALS) have been proposed to destabilize the protein and thereby enhance toxic protein aggregation. In previous studies, denaturation of metallated (holo) hSODs was found to be irreversible, and complicated by the formation of intermolecular disulfide bonds. Here, ALS-associated mutations (E100G, G93A, G85R and A4V) are introduced into a pseudo wild-type background containing no free cysteine residues. The guanidinium chloride-induced denaturation of the holo proteins is generally found to be highly reversible (except for A4V, which tended to aggregate), enabling quantitative analysis of the effects of the mutations on protein stability. Denaturation and renaturation curves were monitored by tryptophan fluorescence, circular dichroism, enzyme activity, chemical cross-linking and analytical sedimentation, as a function of equilibration time and protein concentration. There is strong kinetic hysteresis, with curves requiring exceptionally long times (many days for pseudo wild-type) to reach equilibrium, and evidence for the formation of kinetic and equilibrium intermediate(s), which are more highly populated at lower protein concentrations. The effects of metal dissociation were included in the data fitting. The full protein concentration dependence is best described using a three-state model involving metallated native dimer, metallated monomeric intermediate and unfolded monomers with no bound metals; however, at high protein concentrations the unfolding approaches a two-state transition with metal binding to both the native dimers and unfolded monomers. We show that the E100G, G93A and G85R mutations decrease overall protein stability, largely by decreasing monomer stability with little effect on dimer dissociation. Comparison of the chemical denaturation data with ALS disease characteristics suggests that aggregation of some mutant hSOD may occur through increased population of partially folded states that are less stable than the monomeric intermediate and accessed from the destabilized holo protein.     

Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Evaluation of conformational changes in diabetes‐associated mutation in insulin a chain: A molecular dynamics study

Insulin plays a central role in the regulation of metabolism in humans. Mutations in the insulin gene can impair the folding of its precursor protein, proinsulin, and cause permanent neonatal‐onset diabetes mellitus known as Mutant INS‐gene induced Diabetes of Youth (MIDY) with insulin deficiency. To gain insights into the molecular basis of this diabetes‐associated mutation, we perform molecular dynamics simulations in wild‐type and mutant (Cys^A7 to Tyr or C(A7)Y) insulin A chain in aqueous solutions. The C(A7)Y mutation is one of the identified mutations that impairs the protein folding by substituting the cysteine residue which is required for the disulfide bond formation. A comparative analysis reveals structural differences between the wild‐type and the mutant conformations. The analyzed mutant insulin A chain forms a metastable state with major effects on its N‐terminal region. This suggests that MIDY mutant involves formation of a partially folded intermediate with conformational change in N‐terminal region in A chain that generates flexible N‐terminal domain. This may lead to the abnormal interactions with other proinsulins in the aggregation process. Proteins 2015; 83:662–669. © 2015 Wiley Periodicals, Inc.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the ¹⁹F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the ¹⁹F linewidth and R₂ relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of ¹⁹F probes at additional TTR sites yielded distinct ¹⁹F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The ¹⁹F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.     

Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein

The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal domain and dimerization of the C-terminal domain of CA (CAC), and both domains constitute potential targets for anti-HIV therapy. CAC homodimerization occurs mainly through its second helix, and it is abolished when its sole tryptophan is mutated to alanine. This mutant, CACW40A, resembles a transient monomeric intermediate formed during dimerization. Its tertiary structure is similar to that of the subunits in the dimeric, non-mutated CAC, but the segment corresponding to the second helix samples different conformations. The present study comprises a comprehensive examination of the CACW40A internal dynamics. The results obtained, with movements sampling a wide time regime (from pico- to milliseconds), demonstrate the high flexibility of the whole monomeric protein. The conformational exchange phenomena on the micro-to-millisecond time scale suggest a role for internal motions in the monomer-monomer interactions and, thus, flexibility of the polypeptide chain is likely to contribute to the ability of the protein to adopt different conformational states, depending on the biological environment.     

Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

Formation of fibrous aggregates from a non-native intermediate: the isolated P22 tailspike beta-helix domain.

In the assembly pathway of the trimeric P22 tailspike protein, the protein conformation critical for the partitioning between productive folding and off-pathway aggregation is a monomeric folding intermediate. The central domain of tailspike, a large right-handed parallel beta-helix, is essentially structured in this species. We used the isolated beta-helix domain (Bhx), expressed with a hexahistidine tag, to investigate the mechanism of aggregation without the two terminal domains present in the complete protein. Although Bhx has been shown to fold reversibly at low ionic strength conditions, increased ionic strength induced aggregation with a maximum at urea concentrations corresponding to the midpoint of urea-induced folding transitions. According to size exclusion chromatography, aggregation appeared to proceed via a linear polymerization mechanism. Circular dichroism indicated a secondary structure content of the aggregates similar to that of the native state, but at the same time their tryptophan fluorescence was largely quenched. Microscopic analysis of the aggregates revealed a variety of morphologies; among others, fibrils with fine structure were observed that exhibited bright green birefringence if viewed under cross-polarized light after staining with Congo red. These observations, together with the effects of folding mutations on the aggregation process, indicate the involvement of a partially structured intermediate distinct from both unfolded and native Bhx.     

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of > or =100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12-17 of full-length insulin, was identified as a short peptide that interacts with full-length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near-equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two-step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.     

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Chain assembly intermediate in the biosynthesis of type III procollagen in chick embryo blood vessels

A general mechanism for the assembly of procollagens is proposed from a biosynthetic study of procollagen III. This was shown to proceed by a stepwise process punctuated by disulfide bond formation and an assembly intermediate was recovered. The biosynthesis of type III procollagen in excised chick embryo blood vessels was studied by radioactive labeling for 30 min. Velocity sedimentation under denaturing conditions and purified antibodies specific against bovine amino propeptide III were used to identify and characterize monomeric pro alpha 1 III chains and a type III procollagen intermediate which is interchain disulfide-linked only at the carboxyl end but not at the amino end. The monomeric chains presumably have intrachain disulfide bonds within the propeptides. The monomeric pro alpha 1 III chains were also found when alpha, alpha'-dipyridyl was present during incubation. Pulse-chase experiments show that the monomeric chains and the intermediate are biosynthetic precursors of type III procollagen. Furthermore, it is shown that monomeric pro alpha 1 chains are not triple helical when extracted under nondenaturing conditions. The results indicate that the assembly of pro alpha 1 III chains into type III procollagen starts with the association of the folded carboxyl propeptides and is followed by formation of disulfide bonds between carboxyl propeptides, folding of the triple helix, and formation of disulfide bonds between amino propeptides. All procollagens may follow a similar assembly sequence.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I

In the range of guanidine hydrochloride concentrations from 0.2 to 1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine. Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation. Under some conditions, all the functional properties do not disappear at the same rate: an intermediate species possessing only the kinase activity can be detected. Several arguments suggest that this partly active intermediate has a monomeric structure. These results show that deactivation of aspartokinase-homoserine dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation [Garel, J.-R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions. The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.     

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.