Joseph Horwitz

The major intrinsic protein (MIP) of the bovine lens fiber membrane: Characterization and structure based on cDNA cloning

Synthetic oligonucleotide probes have been used to identify two overlapping cDNA clones that represent the entire coding region of the mRNA for the major intrinsic protein (MIP) of bovine lens cell membrane. Hybridization studies indicate that bovine MIP is encoded by a single-copy gene. The cDNA hybridizes to the rat genome, but MIP mRNA is not detected in rat liver. Analysis of the deduced amino acid sequence provides support for the potential role of MIP as a junctional protein. The structure predicted for MIP suggests that it traverses the lipid bilayer six times with both carboxy and amino termini on the cytoplasmic side, and that it has at least one amphiphilic transmembrane segment, as expected if the protein were to participate in the formation of an aqueous channel.

Disruption of α3 Connexin Gene Leads to Proteolysis and Cataractogenesis in Mice

Abstract Gap junction channels formed by α 3 (Cx46) and α 8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of α 3 connexin in vivo, the α 3 connexin gene was disrupted in mice. Although the absence of α 3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted α 3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell–cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

Subunit Exchange of αA-Crystallin

α-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each α-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant α-crystallin subunits. We labeled αA-crystallin with stilbene iodoacetamide (4-acetamido-4′-((iodoacetyl)amino)stilbene-2,2′-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled αA-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent αA-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min−1 at 37 °C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several αA-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of αA-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function.

Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC59–163) and human alphaB crystallin (ABC68–162), both containing the C‐terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C‐terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C‐terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC59–163 and ABC68–162. A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

LENS ALPHA -CRYSTALLIN : CHAPERONE-LIKE PROPERTIES

Publisher Summary The chapter presents a study on the chaperone-like properties of lens α -crystalline. The most common source for lens α -crystallin has been cow or calf lens. The chapter presents a protocol for preparing calf or cow lens α -crystallin as well as the preparation and purification of recombinant human α B-crystallin. Several assays for the chaperone-like properties of α -crystallin are presented. α -crystallin is one of the abundant structural proteins of the vertebrate eye lens, where it can account for about 40% of the total soluble mass. The a-crystallin family consists of two genes, α A and α B. α B-crystallin has now been found in numerous tissues of the body, such as heart, skeletal muscle, brain, lung, skin, and kidney. α A-crystallin is much less abundant outside the eye lens. α B-crystallin is overexpressed in many degenerative diseases. The biochemical, biophysical, gene regulation, expression, and evolutionary properties of α -crystallin have been studied extensively. Recombinant α -crystallin has properties similar to those of native α -crystallin isolated from eye lens.

Mimicking phosphorylation of αB-crystallin affects its chaperone activity

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Phosphorylation of αB-crystallin alters chaperone function through loss of dimeric substructure

Phosphorylation is the most common posttranslational modification of the α-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of αB-crystallin. Here we show that phosphorylation of αB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse αB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of αB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue.

Association of the Chaperone αB-crystallin with Titin in Heart Muscle

αB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. αB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309–319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of αB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. αB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, αB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between αB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by αB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with αB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

Evidence that α-crystallin prevents non-specific protein aggregation in the intact eye lens

Abstract The ocular lens is a transparent organ comprised of a highly concentrated and highly ordered matrix of structural proteins, called crystallins, which are probably the longest lived proteins of the body. Lens transparency is dependent upon maintenance of the short range order of the crystallin matrix. This transparency must be maintained for decades in the absence of normal protein synthesis or repair capacity. We present evidence here that α-crystallin, one of the major lens proteins, plays a central role in vivo in stabilizing the other crystallins and preventing uncontrolled aggregation of these progressively modified and aging molecules. α-Crystallin has previously been shown to suppress non-specific aggregation of denaturing proteins in simple binary systems through a chaperone-like activity. Our studies using soluble homogenates of monkey lenses demonstrate a strong resistance to heat induced non-specific aggregation when the complete complement of crystallins is present; in contrast, if α-crystallin is selectively removed prior to heating, the remaining crystallins undergo extensive non-specific aggregation as indicated by light scattering. When α-crystallin is present it complexes with denaturing proteins forming a soluble heavy molecular weight (HMW) fraction but no insolubilization is observed, while when α-crystallin is absent there is heavy insolubilization and no HMW formed. When intact monkey lenses were heated it could be demonstrated that soluble HMW was generated. Similar HMW protein appears in vivo in the human lens as a function of age. These findings suggest that the soluble HMW protein present in the human lens is the product of the chaperone-like function of α-crystallin and that under physiological conditions α-crystallin inhibits the uncontrolled aggregation of damaged proteins, thereby preventing the formation of light scattering centers and opacification of the lens.

Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of β-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Lebers congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.