An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development

Khadka, Nawal K.; Timsina, Raju; Mainali, Laxman

doi:10.3390/membranes12050522

Cited by 5 publications

(15 citation statements)

References 69 publications

(130 reference statements)

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“…The average breakthrough force in defect-free membrane regions estimated from three independent experiments is 5.11 ± 1.35 nN. A similar breakthrough force was obtained earlier in αcrystallin-free membrane regions [35]. The elastic nature of the membrane is maintained as indicated by the clear breakthrough events in the representative force curves shown in Figure 5B.…”

Section: Mechanical Properties Of the Membrane-crystallin Systemsupporting

confidence: 85%

“…As shown in Figure 5D, the force curves obtained in the semi-transmembrane defect region (β L -crystallin membrane association region) did not display typical membrane force curves with breakthrough events within the set force threshold limit, suggesting the loss of membrane elasticity. Previously, we have observed similar force curves without breakthrough events within the defined peak force threshold and loss of typical membrane force curve behavior in the α-crystallin-submerged membrane region and the close vicinity of the α-crystallin-associated membrane regions, indicating the loss of membrane elasticity [35].…”

Section: Mechanical Properties Of the Membrane-crystallin Systemsupporting

confidence: 73%

“…Such semi-transmembrane defects might contribute to light scattering and cataract formation, and an increase in the size of the transmembrane defects suggests increased light scattering. Previously, we have investigated the association of α-crystallin with POPC SLM and found that α-crystallin oligomers did not uniformly associate with the membranes; instead clustered as an aggregate in the membrane forming α-crystallin-membrane complexes with increasing size as incubation time increased [35]. The study reported here (Figure 1) shows that β L -crystallin interacts with the membrane forming the semi-transmembrane defects, and with increasing incubation time, the size of such defects increases, suggesting that β L -and α-crystallin modulate with the membrane differently.…”

Section: Interaction Of β L -Crystallin With Membranementioning

confidence: 99%

“…Multiple studies on the interaction of α-crystallin with model and plasma membranes have been performed by our lab [32,[35][36][37][38][39][40] and several others [41][42][43][44][45]. However, knowledge of the interactions of βand γ-crystallin with membranes is limited.…”

Section: Introductionmentioning

confidence: 99%

“…We recently developed the atomic force microscopy (AFM) approach to investigate the high-Chol-containing membranes relevant to eye lens membrane [61,62]. We also developed the AFM approach to investigate the interaction of α-crystallin with the phospholipid membrane and found a strong time-dependent association of α-crystallin to the membrane [35]. Most of the studies on cataract formation are connected with the posttranslational modification (e.g., deamidation.…”

Section: Introductionmentioning

confidence: 99%

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Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy

Khadka,

Hazen,

Haemmerle

et al. 2023

IJMS

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Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.

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Section: Mechanical Properties Of the Membrane-crystallin Systemsupporting

confidence: 85%

Section: Mechanical Properties Of the Membrane-crystallin Systemsupporting

confidence: 73%

Section: Interaction Of β L -Crystallin With Membranementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy

Khadka,

Hazen,

Haemmerle

et al. 2023

IJMS

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Unveiling the structural and functional consequences of the p.D109G pathogenic mutation in human αB-Crystallin responsible for restrictive cardiomyopathy and skeletal myopathy

Hosseini Jafari,

Shahsavani,

Hoshino

et al. 2024

International Journal of Biological Macromolecules

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Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region

Barati,

Rezaei Somee,

Shahsavani

et al. 2024

Sci Rep

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The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

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An AFM Approach Applied in a Study of α-Crystallin Membrane Association: New Insights into Lens Hardening and Presbyopia Development

Cited by 5 publications

References 69 publications

Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy

Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy

Unveiling the structural and functional consequences of the p.D109G pathogenic mutation in human αB-Crystallin responsible for restrictive cardiomyopathy and skeletal myopathy

Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region

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