Degradation of C-terminal truncated alpha A-crystallins by the ubiquitin-proteasome pathway

Abstract

Purpose: Calpain-mediated C-terminal cleavage of alpha 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 alpha A-crystallins.

Methods: Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated alpha A-crystallins were labeled with (125)I, 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 alpha A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering.

Results: Whereas wt alpha A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, alpha A(1-168)-crystallin was resistant to degradation. The susceptibility of alpha A(1-163)-crystallin to degradation was comparable to that of wt alpha A-crystallin. However, alpha A(1-162)-crystallin was much more susceptible than wt alpha A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated alpha 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 alpha A-crystallins revealed that C-terminal truncation of alpha 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 alpha A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of alpha A(1-162) by the UPP may prevent its accumulation in the lens.

FIGURE 1

SDS-PAGE of wt and three truncated forms of αA-crystallin. Lane 1: low-molecular-weight protein standards, the molecular weights were labeled as kDa; lane 2: purified wt αA-crystallin; lane 3: purified truncated αA_1–168-crystallin; lane 4: purified truncated αA_1–163-crystallin; lane 5: purified truncated αA_1–162-crystallin. Twenty micrograms of each purified protein was loaded and the gel was stained with Coomassie blue R-250.

FIGURE 2

Degradation of wt and C-terminal truncated αA-crystallins. The wt and C-terminal truncated αA-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 αA-crystallin. #P < 0.05, ##P < 0.001, in the absence of MG132.

FIGURE 3

Ubiquitin and Ubc4 are essential for degradation of C-terminal truncated αA_1–162-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 αA-crystallin; δ 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.

FIGURE 4

C-terminal truncation induced conformational changes of αA-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.

FIGURE 5

C-terminal truncation alters thermal stability of αA-crystallin. The wt and truncated αA-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.

FIGURE 6

C-terminal truncation alters oligomerization of αA-crystallin. The oligomer sizes of wt and C-terminal truncated αA-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).