HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zhang, X. Articles by Shang, F. Search for Related Content PubMed PubMed Citation Articles by Zhang, X. Articles by Shang, F.

Degradation of C-terminal Truncated A-crystallins by the Ubiquitin–Proteasome Pathway

¹From the Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts; the ²Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; the ³Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
PURPOSE. Calpain-mediated C-terminal cleavage of A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated A-crystallins.

METHODS. Recombinant wild-type (wt) A-crystallin and C-terminal truncated A_1–168-, A_1–163-, and A_1–162-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated A-crystallins were labeled with ¹²⁵I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe Bis-ANS were used to characterize the wt and truncated A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering.

RESULTS. Whereas wt A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, A_1–168-crystallin was resistant to degradation. The susceptibility of A_1–163-crystallin to degradation was comparable to that of wt A-crystallin. However, A_1–162-crystallin was much more susceptible than wt A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated A_1–162-crystallins requires adenosine triphosphate (ATP) and was stimulated by addition of a ubiquitin-conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative mutant of ubiquitin, K6W-Ub, indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wt and C-terminal truncated A-crystallins revealed that C-terminal truncation of A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation.

CONCLUSIONS. The present data demonstrate that C-terminal cleavage of A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of A_1–162 by the UPP may prevent its accumulation in the lens.

Protein aggregation and precipitation is one of the causes of lens opacification. Studies indicate that calpain-mediated cleavage of lens crystallins plays an important role in the aggregation and precipitation of lens proteins.^{8 9 10 11} This unregulated cleavage of crystallins may result in the precipitation of ß-crystallins or cytoskeletal proteins^{12 13 14} or reduction of chaperone activity of -crystallins.¹⁵ Calpains belong to a superfamily of structurally related, calcium-activated cysteine proteases.^{16 17 18} Some calpains are ubiquitously expressed, whereas others are tissue specific.^{17 19 20 21} Studies of various cataract animal models have suggested that Lp82 and calpain-2 may be the major calpains involved in murine cataractogenesis.^{10 11 22 23 24 25 26 27 28}

Both - and ß-crystallins, but not -crystallins, are susceptible to calpain-mediated cleavage.²⁹ Whereas -crystallins are cleaved by calpains at the C terminus,³⁰ ß-crystallins are cleaved closer to the N terminus.³¹ C-terminal cleavage of A-crystallin by calpains can occur at several sites. The major cleaved products of A-crystallin in the lens include A_1–151, A_1–156, A_1–163, and A_1–168.³⁰ A_1–162 can be generated by incubating A-crystallin with m-calpain in vitro,³⁰ but A_1–162 is barely detectable in normal lenses.^{30 32} However, A_1–162 is readily detected in diabetic cataractous lenses.³² The accumulation of truncated A-crystallins in cataractous lenses could result from an increase in production and/or a decrease in degradation of the truncated products by other proteases.

The ubiquitin-proteasome pathway (UPP) is one of the proteolytic systems that selectively degrade modified or damaged proteins. We have demonstrated in previous studies that lens cells (both lens epithelial cells and lens fiber cells) have a fully functional UPP^{33 34 35 36 37 38} and that the UPP preferentially degrades damaged or modified proteins, including oxidized, glutathiolated and thermally denatured proteins.^{37 39 40 41 42} To investigate the role of the UPP in degradation of C-terminal cleaved A-crystallins, we compared the susceptibility of wt and the C-terminal truncated A-crystallins to UPP-mediated degradation.

The data presented indicate that C-terminal truncation of A-crystallin significantly altered its susceptibility to UPP-mediated degradation. Whereas the susceptibility of A_1–163 to UPP-mediated degradation was similar to that of wild-type (wt) A-crystallin, A_1–168 was less susceptible and the A_1–162 more susceptible than wt A-crystallin to UPP-mediated degradation. The rapid degradation of A_1–162 by the UPP may explain why A_1–162-crystallin is barely detectable in the normal lenses. Because the A_1–162-crystallin is less thermally stable and prone to aggregation, the timely degradation of the A_1–162-crystallin may prevent its accumulation and aggregation in the lens.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Construction of C-terminal Truncated A-crystallins
To mimic C-terminal cleavage by calpains, recombinant wt and C-terminal truncated A_1–168, A_1–163, and A_1–162-crystallins were expressed and purified to homogeneity. To construct these truncated A-crystallin, human A-crystallin cDNA in the pAED4 vector⁴³ was used as the template and 5'-ACTCCATGGACGTGACCATCCAG-3' was used as the forward primer for the PCR-based cloning. The reverse primers for A_1–168, A_1–163, and A_1–162 were 5'- ACAGGATCCTTAGGTGGGCTTC-3', 5'-AATGGATCCTTACCGCGACACG-3' and 5'- CATATGTTACGACACGGGGATGG-3', respectively. PCR conditions were as follows: predenaturing at 94°C for 2 minutes, followed by 35 cycles of denaturing at 94°C for 15 seconds, annealing at 63°C for 40 seconds, and extension at 72°C for 40 seconds. PCR products were analyzed on a 1% agarose gel and purified (QIAquick Gel Extraction Kit; Qiagen, Chatsworth, CA). Purified PCR products of wt and truncated A-crystallins were digested with NcoI and inserted into NcoI and BamHI (the BamHI site was blunted by a Klenow fragment of DNA polymerase I) sites of the pET15b vector, to generate pET15b-A, pET15b-A_1–168, pET15b-A_1–163, and pET15b-A_1–162 plasmids. The sequences of these plasmids were verified by DNA sequencing.

Expression and Purification of Recombinant wt and Truncated A-crystallins
The expression plasmids pET15b-A, pET15b-A_1–168, pET15b-A_1–163, and pET15b-A_1–162 were transformed into competent E. coli BL21 (DE3) cells. Growth, induction, and purification of the recombinant proteins were performed essentially as described elsewhere.^{43 44} Briefly, the expression of A-crystallin was induced by adding 1 mM IPTG (isopropyl-ß-d-thiogalactopyranoside) to the culture medium and incubating for 4 hours. The bacteria were collected by centrifugation at 3000g for 10 minutes. The bacterial pellets were lysed in the lysis buffer (50 mM Tris-HCl, 1 mM EDTA [pH 7.6]) by sonication, and the cell lysates were centrifuged at 30,000g for 20 minutes. Whereas >90% of wt A and A_1–168 were in the supernatant, >50% of A_1–163 and A_1–162 were in the pellets. To retrieve A_1–163 and A_1–162, we subjected the pellets to extraction with the lysis buffer containing 0.5% Tween-20 with a brief sonication. The supernatant of Tween-20 extraction was combined with the water-soluble fraction for purification of the recombinant proteins. The supernatants were first fractionated with a DE52 column. Both wt- and C-terminal truncated A-crystallins were eluted in the fractions containing 100 to 200 mM NaCl. After concentration with a centrifugal filter device (Millipore, Bedford, MA), the A-containing fractions were further purified with a Sephacryl S-300 size-exclusion column, with 50 mM Tris-HCl buffer containing 150 mM NaCl (pH. 7.6) as the mobile phase. The concentrations of purified proteins were determined by measuring absorption at 280 nm, with the absorbance coefficients of A 0.1% at 0.742, 0.741, 0.764, and 0.771 for wt A, A_1–168, A_1–163, and A_1–162, respectively, calculated based on the amount of aromatic amino acids.⁴⁵ The purity of wt and truncated proteins was analyzed on 15% SDS-polyacrylamide gels under reducing conditions and stained with Coomassie blue (Brilliant Blue R250; Sigma-Aldrich, St. Louis, MO). To determine the oligomer states of wt and truncated A-crystallins, we analyzed the A-crystallin solutions by size-exclusion chromatography with an inline light-scattering, absorbance, and refractive index detectors.¹ A Sepharose column (6HR 10/30; GE Healthcare, Piscataway, NJ) was connected in line with a UV detector (UV-900; GE Healthcare), a multiangle laser light-scattering detector (Dawn-EOS; Wyatt Technology Corp., Santa Barbara, CA), and a refractive index detector (Optilab-DSP; Wyatt Technology Corp.). Samples were loaded onto the column at a concentration of 1 mg/mL and eluted with 50 mM sodium phosphate buffer containing 100 mM NaCl (pH 7.0).

Study of Conformational Changes
Circular dichroism (CD) spectra were obtained with a circular dichroism spectrometer (Aviv Circular Dichroism Spectrometer model 60 DS; Aviv Associates, Lakewood, NJ). Proteins in 50 mM Tris-HCl buffer (pH 7.6; 0.1 mg/mL) were used. For each CD measurement, five scans were recorded, averaged, and followed by a polynomial fitting program. The CD was expressed in units of deg/cm²/dmol. Fluorescence was measured with a Shimadzu spectrofluorometer (model RF-5301PC; Shimadzu Instruments, Columbia, MD). Tryptophan fluorescence spectra were determined using an excitation wavelength of 295 nm. Bis-ANS fluorescence emission spectra were scanned between 460 and 560 nm with an excitation wavelength of 395 nm. Aliquots of 50 µL of Bis-ANS (5.5 x 10^–5 M stock solution) were added to 1 mL of A-crystallin solution (0.1 mg/mL in 50 mM Tris-HCl buffer; pH 7.6) until saturation was reached. The samples were then incubated for 10 minutes at room temperature before the fluorescence spectra were determined.

Thermal Stability Measurements
Thermal stability was studied by time-dependent changes in light-scattering at 400 nm using a fluorescence spectrophotometer (excitation and emission wavelengths were set at 400 nm) when wt or truncated A-crystallins (in 50 mM sodium phosphate buffer; pH 7.6) were incubated at 65°C. The protein melting temperature (Tm) was determined with a VP-capillary differential scanning calorimeter (MicroCal, Northampton, MA). The protein concentration was 1 mg/mL in 50 mM Tris-HCl buffer (pH 7.6). The starting temperature was 10°C, and the final temperature was 110°C. The scanning rate is 100°C/hour. Commercial software (Origin; MicroCal) was used to calculate the Tm of the proteins.

Preparation of Lens Fiber Lysate and Reticulocyte Lysate
Fresh calf eyes (within 6 hours of death) were purchased from a local meat-packing company, and the outer layers of lens cortex was homogenized with 50 mM Tris-HCl buffer containing 1 mM dithiothreitol (DTT; pH 7.6). After centrifugation at 100,000g for 10 minutes, the supernatant was used as the source of the UPP for degradation assays. Rabbit reticulocytes were purchased from Green Hectares Company (Oregon, WI). After three washes with PBS, the packed recticulocytes were lysed with an equal volume of 10 mM Tris-HCl containing 1 mM DTT (pH 7.6) as previously described.³⁹ After centrifugation at 50,000g for 60 minutes, the supernatant was used as the source of the UPP for degradation assays.

Proteolytic Degradation Assay and Statistical Analysis
Both wt and C-terminal truncated A-crystallins were labeled with ¹²⁵I by the chloramine T method.³⁹ Free ¹²⁵I and small peptides were removed by Sephadex G25 desalting columns, and labeled proteins were concentrated with microconcentrators (Centricon 10; Amicon, Beverly, MA). The specific activity of ¹²⁵I-labeled proteins ranged from 0.2 to 0.5 µCi/µg. An approximately equal amount of substrate was used for the degradation assay (100 ng/assay). Degradation of the wt and truncated A-crystallins was determined as described by Huang et al.,⁴⁶ but we used bovine lens fiber lysate or rabbit reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Briefly, the proteolysis reaction mixture, in a final volume of 25 µL, contained 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 1 mM DTT, and 15 µL lens fiber lysate (150mg/mL protein), or reticulocyte lysate (300 mg/mL protein). For determination of adenosine triphosphate (ATP)- and Ubc4-dependent proteolysis, 2 mM ATP, 10 mM creatine phosphate, 6 µg creatine phosphokinase, and 0.4 µg recombinant Ubc4 was included in the assay. The latter was expressed and purified essentially as described by Wing and Jain.⁴⁷ Pilot experiments suggested that there is sufficient free ubiquitin in lens fiber lysate and reticulocyte lysate; therefore, no exogenous ubiquitin was added in these assays. Degradation was initiated by addition of 4 to 10 x 10⁴ cpm of ¹²⁵I-labeled A-crystallins, and the reaction mixtures were incubated at 37°C for 2 hours. The reactions were terminated by addition of 200 µL of ice-cold 10 mg/mL bovine serum albumin, immediately followed by 50 µL of 100% TCA (yielding a final concentration of 18.2% TCA), after which the samples were left on ice for 10 minutes. The extent of degradation was determined as the amount of TCA-soluble ¹²⁵I-labeled fragments of A-crystallin. The total TCA-insoluble count at time 0 was defined as 100%.

In pilot experiments, the percentage of degradation was proportional to the incubation time for the first 2 hours. The percentage of degradation during the 2-hour incubation reflected the susceptibility to degradation. Since the substrate was not saturating and the degradation was expressed as a percentage of the labeled substrate that was degraded during the 2 hours of incubation, the susceptibility to degradation is not affected by variation in the molar amount of the labeled substrates, at least under these experimental conditions.

The degradation observed with the addition of ATP and Ubc4 is referred to as total degradation, whereas the difference between total degradation and degradation without addition of ATP and Ubc4 is denoted as ATP-stimulated degradation. All experiments were performed in triplicate and repeated two to four times. For statistical analysis, data from several experiments were pooled and analyzed using Student's t-test.

To determine whether the degradation measured in this assay was proteasome-dependent, MG132, a proteasome inhibitor, was added to the system at a final concentration of 24 µM. To determine ubiquitin-dependent degradation, the proteolytic assay was also performed with addition of 2 µg of a dominant negative mutant ubiquitin (K6W-Ub), which specifically inhibits ubiquitin-dependent proteolysis.⁴⁸ This results in 1:1 ratio of endogenous wt ubiquitin and added K6W-Ub.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Expression and Purification of wt and C-terminal Truncated A-crystallins
The wt and truncated A-crystallins were expressed in E. coli BL21 (DE3) and expression of recombinant proteins was monitored by SDS-PAGE. The wt and A_1–168-crystallins were mainly found in the water-soluble fraction, whereas >50% of A_1–163 and A_1–162-crystallins were found in the water-insoluble fraction. The wt and A_1–168-crystallin were purified from the water-soluble fraction to near homogeneity (95%). The A_1–163 and A_1–162-crystallins were first retrieved from the water-insoluble fraction with a 50 mM Tris-HCl buffer containing 0.5% Tween-20 (pH. 7.6), and the proteins were then purified to near homogeneity (95%) using the same procedures as used for the purification of wt protein. As shown in Figure 1 , the purified proteins migrated predominantly as a single 20-kDa band on SDS-PAGE. The differences in migration due to C-terminal truncations were clearly detectable. These differences in migration may reflect not only the changes in molecular sizes of these truncated proteins, but also the changes in charges of the truncated proteins.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. SDS-PAGE of wt and three truncated forms of A-crystallin. Lane 1: low-molecular-weight protein standards, the molecular weights were labeled as kDa; lane 2: purified wt A-crystallin; lane 3: purified truncated A1–168-crystallin; lane 4: purified truncated A1–163-crystallin; lane 5: purified truncated A1–162-crystallin. Twenty micrograms of each purified protein was loaded and the gel was stained with Coomassie blue R-250.

Degradation of C-terminal Truncated A-crystallins by the UPP

View larger version (12K): [in this window] [in a new window]   FIGURE 2. Degradation of wt and C-terminal truncated A-crystallins. The wt and C-terminal truncated A-crystallins were labeled with 125I and the ATP-dependent degradation assay was performed using lens fiber cell lysate (A) or rabbit reticulocyte lysate (B) as the source of UPP components, with or without addition of the proteasome inhibitor MG132. *P < 0.05, **P < 0.001, compared with the degradation of wt A-crystallin. #P < 0.05, ##P < 0.001, in the absence of MG132.

The results above indicate that A_1–162, but not A_1–168 and A_1–163, is more susceptible than wt A-crystallin to UPP-mediated degradation. To determine further the involvement of ubiquitin and ubiquitination in the selective degradation of A_1–162, we assessed the effect of a dominant negative mutant ubiquitin (K6W-ubiquitin).⁴⁸ As shown in Figure 3A , addition of K6W-ubiquitin not only reduced the degradation of wt and A_1–162-crystallins, but it also diminished the difference in degradation between wt and A_1–162-crystallins (Fig. 3A) . These data further demonstrated that A_1–162-crystallin was preferentially degraded in a ubiquitin-dependent manner. To further corroborate the requirement of ubiquitination in degradation of wt and A_1–162-crystallins, we determined the effects of a ubiquitin-conjugating enzyme, Ubc4, on degradation of wt and A_1–162-crystallins. Previous work demonstrated that Ubc4 plays a role in degradation of abnormal proteins and that the level of Ubc4 is limiting in reticulocytes.⁴¹ The difference in degradation between wt and A_1–162-crystallins was marginal when Ubc4 was not added (Fig 3B) . Addition of Ubc4 only slightly increased the degradation of wt A-crystallin. In contrast, addition of Ubc4 significantly increased the degradation of A_1–162-crystallin (Fig. 3B) . The data further confirm that the UPP plays a role in the selective degradation of C-terminal truncated A_1–162-crystallin.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Ubiquitin and Ubc4 are essential for degradation of C-terminal truncated A1–162-crystallin. (A) The wt ubiquitin or dominant-negative mutant K6W-ubiquitin (320 ng/µL; final concentration) was added to the degradation system. (B) The degradation was performed in the presence or absence of 20 ng/µL recombinant Ubc4. *P < 0.05, **P < 0.001 compared with the degradation of wt A-crystallin; indicates P < 0.01 when compared with the degradation with supplementation of wt ubiquitin; #P < 0.05 when compared with the degradation in the absence of Ubc4.

Effects of C-terminal Truncation on the Secondary and Tertiary Structures of A-crystallin

The far UV CD spectrum reflects the secondary structure of a protein. Figure 4A shows that C-terminal truncations of A-crystallin slightly altered the CD spectra and that different truncations had different effects on the CD spectrum. The effects of C-terminal truncation on contents of -helix, ß-sheet, ß-turn, and random coil are summarized in Table 1 . In general, C-terminal truncation did not significantly alter the secondary structures. C-terminal truncated A-crystallins had a moderate increase in contents of ß-sheet and a decrease in contents of ß-turn. The C-terminal truncations had no significant effect on the contents of -helix and random coil (Table 1) . For accurate determination of the secondary structure of a protein, the CD spectrum should be scanned from 260 to 180 nm.⁵⁰ For comparative purposes, the far UV CD spectrum in this study was scanned from 260 to 200 nm. This may affect the accuracy of the estimated contents of -helix, ß-sheet, ß-turn, and random coil of these proteins, but it is sufficient for evaluating the conformational changes of the truncated proteins.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. C-terminal truncation induced conformational changes of A-crystallin. (A) Far UV CD spectra. Protein concentration was 0.1 mg/mL in 50 mM Tris-HCl buffer (pH 7.6), and cellpath length was 1 mm. The spectra were an average of five scans smoothed by a polynomial-fitting program. (B) Tryptophan fluorescence. Protein concentration was 0.1 mg/mL in 50 mM Tris-HCl buffer (pH7.6). Excitation wavelength was 295 nm. (C) Surface hydrophobicity. Bis-ANS was added to protein solutions (0.1 mg/mL in 50 mM Tris-HCl buffer; pH 7.6) to a final concentration of 15 µM. Fluorescence was measured at room temperature, with an excitation wavelength of 395 nm.

View this table: [in this window] [in a new window]   TABLE 1. Effect of C-terminal Truncation on the Secondary Structures of A-crystallin

Conformational changes are often associated with changes in the surface hydrophobicity. Bis-ANS (a hydrophobic probe) is nonfluorescent in aqueous solution and becomes fluorescent when it binds to the hydrophobic residues on the surface of a molecule. As shown in Figure 4C , A_1–162-crystallin exhibited an increase in Bis-ANS fluorescence intensity, indicating an increase in hydrophobicity. In contrast, A_1–163- and A_1–168-crystallins showed a decrease in Bis-ANS fluorescence, indicating a decrease in hydrophobicity.

Effect of C-terminal Truncation on the Thermal Stability of A-crystallin
As a member of the small heat shock protein family, A-crystallin is thermally stable and is capable of preventing heat-induced aggregation and precipitation of other proteins.^{5 6} To characterize further the structural changes associated with C-terminal truncated A-crystallins, we compared the thermal stability of wt and truncated A-crystallins. As shown in Figure 5 , A_1–168-crystallin was more thermally stable than was wt A-crystallin. Incubation of wt A-crystallin at 65°C resulted in a slight increase in light-scattering, but incubating A_1–168-crystallin under the same condition did not cause detectable changes in light-scattering. A_1–163-crystallin was also more stable than wt A-crystallin, but not as stable as A_1–168-crystallin. However, A₁-₁₆₂-crystallin was most susceptible to heat-induced aggregation. When incubated at 65°C, A_1–162-crystallin began to aggregate as early as 5 minutes. The altered thermal stabilities of the C-terminal truncated A-crystallins were confirmed by differential scanning calorimetry analysis. The Tm of wt A-crystallin was 59.65 ± 0.14°C. The Tm of A_1–168-, A_1–163-, and A_1–162-crystallins were 64.06 ± 0.09°C, 60.44 ± 0.06°C, and 57.65 ± 0.16°C, respectively. These data indicate that the C-terminus of A-crystallin plays a role in determining thermal stability of A-crystallin.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. C-terminal truncation alters thermal stability of A-crystallin. The wt and truncated A-crystallins in 50 mM sodium phosphate buffer (0.1 mg/mL) were incubated at 65°C and heat-induced light-scattering was detected with a spectrofluorometer. Both the emission and the excitation wavelengths were set at 400 nm.

Effect of C-terminal Truncation on the Oligomerization of A-crystallin

View larger version (14K): [in this window] [in a new window]   FIGURE 6. C-terminal truncation alters oligomerization of A-crystallin. The oligomer sizes of wt and C-terminal truncated A-crystallins in sodium phosphate buffer containing 100 mM NaCl were determined by size exclusion chromatography coupled with a multiangle light-scattering detector. The lines formed by circles represented the molecular weight obtained as a function of the elution volumes (left ordinate).

	Discussion

Top Abstract Materials and Methods Results Discussion References

Levels of calpain-cleaved proteins are determined by the rates of production and the rates of clearance by other proteases. In the lens, various proteases, such as aminopeptidases,^{57 58 59} trypsin-like proteases,^{60 61} and the UPP^{34 35 37 38 62 63 64 65 66} may play a role in the clearance of calpain-cleaved proteins. Among these proteases, the UPP has been demonstrated to be involved in degradation of modified or damaged proteins, including oxidized, denatured, glutathiolated, and mutant proteins.^{35 37 41 42 46 62 67 68} Therefore, we hypothesized that the UPP also plays a role in degradation of calpain-cleaved proteins. To test this hypothesis, recombinant C-terminal truncated A-crystallins were used as model substrates for the UPP in lens fiber and reticulocyte lysates. We found that A_1–162 was more susceptible than wt A-crystallin to degradation by the UPP in both lens fiber and reticulocyte lysates. Whereas A_1–163 was degraded similar to wt A, A_1–168 was less susceptible to UPP-mediated degradation.

Consistent with the long lives of lens proteins, only a small fraction of the labeled substrates were degraded during the 2 hours of incubation with lens fiber lysate. Whereas 0.91% ± 0.43% of wt A-crystallin was degraded, 3.74% ± 1.87% of A_1–162-crystallin was degraded during this period. Although the difference in susceptibility of wt and C-terminal truncated A-crystallins to degradation is not dramatic in a biochemical sense, this difference could have physiological significance. For example, 1% degradation of the substrate during the 2 hours of incubation means the protein has a half-life of 100 hours. If the degradation increases to 4%, it indicates that the half-life of the protein decreases to 25 hours. Because the chronic accumulation and precipitation of damaged proteins is associated with cataract, a three- to four-fold change in the half-live of a protein could make a significant difference in composition, structure, and optical functions of the lens over a lifetime.

A_1–162 is the major product of m-calpain-cleaved A-crystallin, and it has been detected in diabetic cataractous lenses, but not in normal lens.³² The rapid degradation of A_1–162 by the UPP may be a reason for the absence of A_1–162 in normal lenses. The accumulation of A_1–162 in diabetic cataract lenses could be caused by enhanced m-calpain activity and/or by reduced UPP activity, or both. Since A_1–162 is less thermally stable, the rapid degradation by the UPP prevents it from accumulation and aggregation under stress conditions.

The data also indicate that not all C-terminal truncated A-crystallins are selectively degraded by the UPP. For example, A_1–168 is less susceptible than wt A-crystallin to UPP-mediated degradation, and A_1–163 is degraded similarly to wt A-crystallin. The relative resistance to UPP-mediated degradation of these two C-terminal truncated A-crystallins is consistent with their presence in normal lenses.^{30 32} It appears that some of the calpain-cleaved A-crystallins, such as A_1–168, have to be further cleaved by another protease or peptidases, to be recognized and degraded by the UPP. Consistent with this hypothesis, many of the cleaved fragments of lens proteins, particularly ß-crystallins, do not match the calpain-cleavage sites,⁶⁹ which indicates that calpain-cleaved products are readily trimmed by other proteases or peptidases.

The different percentages of substrates degraded under these conditions may reflect the different proportion of degradation-prone conformers among the comparison groups. As indicated by the multiangle light-scattering analysis (Fig. 6) , the molecular masses of the oligomers of these A-crystallins are not uniform, indicating heterogeneity of conformation in each of these A-crystallins. These data indicate that, whereas A_1–162 tends to adapt a degradation-prone conformer, A_1–168 tends to adapt a degradation-resistant one.

In an attempt to identify the signals for UPP-mediated degradation, we compared the secondary and tertiary structures of wt and different C-terminal truncated A-crystallins. C-terminal truncations of A-crystallin resulted in only subtle changes in the CD spectrum, indicating that the C-terminal truncation did not significantly alter the secondary structures. C-terminal truncation also altered tryptophan fluorescence and surface hydrophobicity. Consistent with conformational changes, C-terminal truncation changed oligomeric states and thermal stabilities of A-crystallin. Whereas A_1–168 and A_1–162 formed slightly smaller sizes of oligomers, A_1–163 tended to form large aggregates. Among these biochemical–biophysical changes, surface hydrophobicity and thermal stabilities of these truncated A-crystallins correlated with the susceptibilities to UPP-mediated degradation, which indicates that the thermal stability and surface hydrophobicity of a protein, but not the changes in CD spectrum, tryptophan fluorescence or oligomeric states, can predict the susceptibility to UPP-mediated degradation. However, we cannot rule out the possibility that the subtle changes in secondary, tertiary and/or quaternary structures may contribute to the changes in surface hydrophobicity and thermal stabilities of the truncated crystallins.

The correlation between thermal stability and susceptibility to UPP-mediated degradation indicate that the residues that are recognized by the UPP also play a role in maintaining the thermal stability of a protein. The positive correlation between thermal vulnerability and susceptibility to degradation may be a mechanism for the selective degradation of proteins that are in unstable conformations. This mechanism may play an essential role in preventing the accumulation and aggregation of thermal labile proteins. Hence, this protein quality-control mechanism may be essential for maintaining the transparency of the lens.

The susceptibilities of the wt and C-terminal truncated A-crystallins were determined in this study by using purified proteins. In the lens, C-terminal truncated A-crystallins may form complexes with wt A-crystallin, B-crystallin, or other small heat shock proteins. It remains to be determined whether the C-terminal truncated A-crystallins in complexes with wt -crystallins are degraded in the same manner as in the isolated form. We will determine the effect of wt -crystallins on degradation of C-terminal truncated A-crystallins in future studies.

	Footnotes

 
Supported by National Eye Institute Grants EY11717, EY13250, EY13968 and EY14183, and USDA CRIS 1950-51000-060-01A. AF is a recipient of a fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/19,039/2004).

Submitted for publication February 14, 2007; revised April 20 and May 1, 2007; accepted June 12, 2007.

Disclosure: X. Zhang, None; E.J. Dudek, None; B. Liu, None; L. Ding, None; A.F. Fernandes, None; J.J. Liang, None; J. Horwitz, None; A. Taylor, None; F. Shang, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Fu Shang, USDA Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111; fu.shang{at}tufts.edu.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Horwitz J. Alpha-crystallin. Exp Eye Res. 2003;76:145–153.[CrossRef][ISI][Medline][Order article via Infotrieve]
2. McDevitt DS, Hawkins JW, Jaworski CJ, Piatigorsky J. Isolation and partial characterization of the human alpha a-crystallin gene. Exp Eye Res. 1986;43:285–291.[CrossRef][ISI][Medline][Order article via Infotrieve]
3. Dubin RA, Ally AH, Chung S, Piatigorsky J. Human alpha b-crystallin gene and preferential promoter function in lens. Genomics. 1990;7:594–601.[CrossRef][ISI][Medline][Order article via Infotrieve]
4. Piatigorsky J. Molecular biology: recent studies on enzyme/crystallins and alpha-crystallin gene expression. Exp Eye Res. 1990;50:725–728.[CrossRef][ISI][Medline][Order article via Infotrieve]
5. Horwitz J. A-crystallin can function as a molecular chaperone. Proc Natl Acad Sci USA. 1992;89:10449–10453.[Abstract/Free Full Text]
6. Reddy GB, Das KP, Petrash JM, Surewicz WK. Temperature-dependent chaperone activity and structural properties of human alphaA- and alphaB-crystallins. J Biol Chem. 2000;275:4565–4570.[Abstract/Free Full Text]
7. Boyle DL, Takemoto L, Brady JP, Wawrousek EF. Morphological characterization of the alpha A- and alpha B-crystallin double knockout mouse lens. BMC Ophthalmol. 2003;3:3.[CrossRef][Medline][Order article via Infotrieve]
8. Azuma M, David LL, Shearer TR. Cysteine protease inhibitor e64 reduces the rate of formation of selenite cataract in the whole animal. Curr Eye Res. 1991;10:657–666.[ISI][Medline][Order article via Infotrieve]
9. Azuma M, Fukiage C, David LL, Shearer TR. Activation of calpain in lens: a review and proposed mechanism. Exp Eye Res. 1997;64:529–538.[CrossRef][ISI][Medline][Order article via Infotrieve]
10. Qian W, Shichi H. Cataract formation by a semiquinone metabolite of acetaminophen in mice: possible involvement of ca(2+) and calpain activation. Exp Eye Res. 2000;71:567–574.[CrossRef][ISI][Medline][Order article via Infotrieve]
11. Nakamura Y, Fukiage C, Shih M, et al. Contribution of calpain lp82-induced proteolysis to experimental cataractogenesis in mice. Invest Ophthalmol Vis Sci. 2000;41:1460–1466.[Abstract/Free Full Text]
12. David LL, Dickey BM, Shearer TR. Origin of urea soluble protein in the selenite cataract: role of ß-crystallin proteolysis and calpain ii. Invest Ophthalmol Vis Sci. 1987;28:1148–1156.[Abstract/Free Full Text]
13. Yoshida H, Murachi T, Tsukahara I. Degradation of actin and vimentin by calpain II, a ca2+-dependent cysteine proteinase, in bovine lens. FEBS Lett. 1984;170:259–262.[CrossRef][ISI][Medline][Order article via Infotrieve]
14. Shih M, David LL, Lampi KJ, et al. Proteolysis by m-calpain enhances in vitro light scattering by crystallins from human and bovine lenses. Curr Eye Res. 2001;22:458–469.[CrossRef][ISI][Medline][Order article via Infotrieve]
15. Kelley MJ, David LL, Iwasaki N, Wright J, Shearer TR. A-crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract. J Biol Chem. 1993;268:18844–18849.[Abstract/Free Full Text]
16. Croall DE, DeMartino GN. Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev. 1991;71:813–847.[Free Full Text]
17. Biswas S, Harris F, Dennison S, Singh J, Phoenix DA. Calpains: targets of cataract prevention?. Trends Mol Med. 2004;10:78–84.[CrossRef][ISI][Medline][Order article via Infotrieve]
18. Sorimachi H, Ishiura S, Suzuki K. Structure and physiological function of calpains. Biochem J. 1997;328:721–732.[ISI][Medline][Order article via Infotrieve]
19. Sorimachi H, Imajoh-Ohmi S, Emori Y, et al. Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types: specific expression of the mRNA in skeletal muscle. J Biol Chem. 1989;264:20106–20111.[Abstract/Free Full Text]
20. Fukiage C, Nakajima E, Ma H, Azuma M, Shearer TR. Characterization and regulation of lens-specific calpain lp82. J Biol Chem. 2002;277:20678–20685.[Abstract/Free Full Text]
21. Shih M, Ma H, Nakajima E, et al. Biochemical properties of lens-specific calpain lp85. Exp Eye Res. 2006;82:146–152.[CrossRef][ISI][Medline][Order article via Infotrieve]
22. Azuma M, Shearer TR. Involvement of calpain in diamide induced cataract in cultured lenses. FEBS Lett. 1992;307:313–317.[CrossRef][ISI][Medline][Order article via Infotrieve]
23. Hightower KR, David LL, Shearer TR. Regional distribution of free calcium in selenite cataract: relation to calpain II. Invest Ophthalmol Vis Sci. 1987;28:1433–1436.[Abstract/Free Full Text]
24. David LL, Wright JW, Shearer TR. Calpain II induced insolublization of lens ß-crystallin polypeptides may induce cataract. Biochim Biophys Acta. 1992;1139:210–216.[Medline][Order article via Infotrieve]
25. Sakamoto-Mizutani K, Fukiage C, Tamada Y, Azuma M, Shearer TR. Contribution of ubiquitous calpains to cataractogenesis in the spontaneous diabetic wbn/kob rat. Exp Eye Res. 2002;75:611–617.[CrossRef][ISI][Medline][Order article via Infotrieve]
26. Shearer TR, Shih M, Azuma M, David LL. Precipitation of crystallins from young rat lens by endogenous calpain. Exp Eye Res. 1995;61:141–150.[CrossRef][ISI][Medline][Order article via Infotrieve]
27. Shearer TR, Shih M, Mizuno T, David LL. Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species. Curr Eye Res. 1996;15:860–868.[ISI][Medline][Order article via Infotrieve]
28. Tamada Y, Fukiage C, Mizutani K, et al. Calpain inhibitor, sja6017, reduces the rate of formation of selenite cataract in rats. Curr Eye Res. 2001;22:280–285.[CrossRef][ISI][Medline][Order article via Infotrieve]
29. David LL, Shearer TR. Purification of calpain ii from rat lens and determination of endogenous substrates. Exp Eye Res. 1986;42:227–238.[CrossRef][ISI][Medline][Order article via Infotrieve]
30. Ueda Y, Fukiage C, Shih M, Shearer TR, David LL. Mass measurements of c-terminally truncated alpha-crystallins from two-dimensional gels identify lp82 as a major endopeptidase in rat lens. Mol Cell Proteomics. 2002;1:357–365.[Abstract/Free Full Text]
31. David LL, Shearer TR, Shih M. Sequence analysis of lens beta-crystallins suggests involvement of calpain in cataract formation. J Biol Chem. 1993;268:1937–1940.[Abstract/Free Full Text]
32. Thampi P, Hassan A, Smith JB, Abraham EC. Enhanced c-terminal truncation of A- and B-crystallins in diabetic lenses. Invest Ophthalmol Vis Sci. 2002;43:3265–3272.[Abstract/Free Full Text]
33. Shang F, Deng G, Obin M, et al. Ubiquitin-activating enzyme (e1) isoforms in lens epithelial cells: origin of translation, e2 specificity and cellular localization determined with novel site-specific antibodies. Exp Eye Res. 2001;73:827–836.[CrossRef][ISI][Medline][Order article via Infotrieve]
34. Shang F, Gong X, McAvoy JW, et al. Ubiquitin-dependent pathway is up-regulated in differentiating lens cells. Exp Eye Res. 1999;68:179–192.[CrossRef][ISI][Medline][Order article via Infotrieve]
35. Shang F, Gong X, Palmer HJ, Nowell TR, Taylor A. Age-related decline in ubiquitin conjugation in response to oxidative stress in the lens. Exp Eye Res. 1997;64:21–30.[CrossRef][ISI][Medline][Order article via Infotrieve]
36. Shang F, Gong X, Taylor A. Activity of ubiquitin dependent pathway in response to oxidative stress: ubiquitin activating enzyme (e1) is transiently upregulated. J Biol Chem. 1997;272:23086–23093.[Abstract/Free Full Text]
37. Shang F, Nowell TR, Jr, Taylor A. Removal of oxidatively damaged proteins from lens cells by the ubiquitin-proteasome pathway. Exp Eye Res. 2001;73:229–238.[CrossRef][ISI][Medline][Order article via Infotrieve]
38. Pereira P, Shang F, Girão H, Taylor A. Lens fibers have a fully functional ubiquitin-proteasome pathway. Exp Eye Res. 2003;76:623–631.[CrossRef][ISI][Medline][Order article via Infotrieve]
39. Shang F, Huang L, Taylor A. Degradation of native and oxidized beta- and gamma-crystallins using bovine lens epithelial cell and rabbit reticulocyte extracts. Curr Eye Res. 1994;13:423–431.[ISI][Medline][Order article via Infotrieve]
40. Dudek EJ, Shang F, Valverde P, et al. Selectivity of the ubiquitin pathway for oxidatively modified proteins: relevance to protein precipitation diseases. FASEB J. 2005;19:1707–1709.[Abstract/Free Full Text]
41. Marques C, Guo W, Pereira P, et al. The triage of damaged proteins: degradation by the ubiquitin-proteasome pathway or repair by molecular chaperones. FASEB J. 2006;20:741–743.[Abstract/Free Full Text]
42. Zetterberg M, Zhang X, Taylor A, et al. Glutathiolation enhances the degradation of C-crystallin in lens and reticulocyte lysates, partially via the ubiquitin-proteasome pathway. Invest Ophthalmol Vis Sci. 2006;47:3467–3473.[Abstract/Free Full Text]
43. Sun TX, Das BK, Liang JJ. Conformational and functional differences between recombinant human lens A- and B-crystallin. J Biol Chem. 1997;272:6220–6225.[Abstract/Free Full Text]
44. Sun TX, Liang JJ. Intermolecular exchange and stabilization of recombinant human alphaA- and alphaB-crystallin. J Biol Chem. 1998;273:286–290.[Abstract/Free Full Text]
45. Mach H, Middaugh CR, Lewis RV. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992;200:74–80.[CrossRef][ISI][Medline][Order article via Infotrieve]
46. Huang LL, Shang F, Nowell TR, Jr, Taylor A. Degradation of differentially oxidized -crystallins in bovine lens epithelial cells. Exp Eye Res. 1995;61:45–54.[CrossRef][ISI][Medline][Order article via Infotrieve]
47. Wing SS, Jain P. Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (e2(17)kb) highly expressed in rat testis. Biochem J. 1995;305(Pt 1)125–132.[ISI][Medline][Order article via Infotrieve]
48. Shang F, Deng G, Liu Q, et al. Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation. J Biol Chem. 2005;280:20365–20374.[Abstract/Free Full Text]
49. Yang JT, Wu CS, Martinez HM. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269.[ISI][Medline][Order article via Infotrieve]
50. Bloemendal M, Toumadje A, Johnson WC, Jr. Bovine lens crystallins do contain helical structure: a circular dichroism study. Biochim Biophys Acta. 1999;1432:234–238.[CrossRef][Medline][Order article via Infotrieve]
51. Kosinski-Collins MS, King J. In vitro unfolding, refolding, and polymerization of human D crystallin, a protein involved in cataract formation. Protein Sci. 2003;12:480–490.[Abstract/Free Full Text]
52. Shaeffer JR. Monoubiquitinated alpha globin is an intermediate in the atp-dependent proteolysis of alpha globin. J Biol Chem. 1994;269:22205–22210.[Abstract/Free Full Text]
53. Shaeffer JR. Atp-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent. J Biol Chem. 1988;263:13663–13669.[Abstract/Free Full Text]
54. Takeuchi N, Ouchida A, Kamei A. C-terminal truncation of alpha-crystallin in hereditary cataractous rat lens. Biol Pharm Bull. 2004;27:308–314.[CrossRef][ISI][Medline]