Aggregation in α-Crystallins

The Alpha Crystallins are ~20 kDa small heat shock proteins (sHSP) that play several important roles in the eye.  There are two expressed forms of Alpha Crystallin, Alpha Crystallin A (aXA) and Alpha Crystallin B (aXB). Our lab will be focusing on aXB.  Alpha Crystallin B is well conserved and plays two important and overlapping roles. First, it is a small heat shock protein that prevents misfolded proteins from aggregating in the lens of the eye. This delays or prevents the formation of cataracts, the number one cause of blindness in the world.  Second, the protein takes part in a diverse oligomerization with other crystallins in the lens. This oligomerization is mediated by several protein-protein interaction surfaces in alpha crystallin. The Eisenberg Lab recently solved the crystal structure of most of this protein that showed these interation surfaces, and hinted at implications of its domain swapped C-terminal strand.

With leadership from post-doc Dr. Jim Hebda, our lab has focused on two important questions. First, what is the mechanism of chaperone function. To this end we will study model aggregation systems such as the insulin B-chain. Second, how does each of protein--protein interface contribute to oligomer stability and chaperone function. We will study the oligomerization of the alpha crystallins and the importance of each of the protein-protein interfaces by a variety of techniques. Our work studying the mechanism of chaperone function will inform the design and interpretation of results as we probe the importance of each oligomerization interface.

Interaction of α-Crystallin B in Functional Oligomerization - R.Cabrejo '14

Biophysical Journal, 2014, Vol.106 (2) DOI 10.1016/j.bpj.2013.11.2666

Alpha-Crystallin-B (aXB) is a small heat shock protein found mainly in the lens of the eye. There it serves two purposes: acting as a chaperone to prevent the misfolding of the other protein and contributing to the high protein concentrations required to focus light. When aXB chaperone function fails cataract formation begins. Understanding the dependence of the mechanism of aXB chaperone function on its oligomerization state will aid in the delay or prevention of cataracts. Oligomerization of alpha-crystallinB is pH dependent. We present a mechanism for the chaperone function of aXB using insulin as a model system in which it is possible to induce aggregation. The relationship between oligomerization and chaperone function is tested by measuring the dependence of the onset of light scattering insulin aggregates on the oligomerization state of the aXB. Different oligomerization states can be induced by pre-incubating aXB at a range of pHs. Attenuated chaperone function is observed for aXB pre-incubated at elevated pH even when the assay itself takes place at pH 7. These results are consistent with a model in which aXB is kinetically trapped into higher oligomer states at high pH and unable to return quickly to its functional equilibrium state upon dilution to neutral pH. We will also test the generality of this model by extending our studies to Gamma-Crystallin. Finally, we will measure the contribution of the C-terminal strand to chaperone function and oligomerization using fluorescence resonance energy transfer (FRET). Observation of pH dependent dynamics of the C-terminal strand will help to distinguish between strand-exchanged and dynamic states of the C-terminus, allowing its contribution to oligomerization and chaperone function to be probed.

Characterizing Palindromic Strand Exchange in α-Crystallin Oligomerization - A. Pearlman '13

Biophysical Journal, 2013, Vol. 104, p394a;  DOI:

Alpha-crystallin, a member of the small heat shock protein (sHSP) class, is expressed in eye lens, muscle, and brain tissue where it contributes to homeostasis by chaperoning and thus preventing the pathogenic aggregation of damaged or unfolded proteins. As a major cytoplasmic component in the eye lens, alpha-crystallin is further essential for producing a high index of refraction. Despite its high concentrations, alpha-crystallin does not itself aggregate or crystallize, a property facilitated by its populating dynamic, polydisperse assemblies. Oligomerization in this manner is a common feature of the sHSPs and is likely connected to their chaperone function.

Structural studies1 suggest that one mechanism of polydispersity may be the ability of a palindromic sequence centered on the sHSP IXI motif in the alphaB-crystallin (αB) isoform c-terminus to bind bidirectionally to other monomers. Bidirectional strand exchanges would result in heterogeneous oligomeric structures while maintaining near identical residue interactions.

Modeling Disfunction in α-Crystallin-J.Santos '12


Alpha Crystallin is the major protein component of the human lens and plays an important role in the prevention of cataracts. α -Crystallin (α X) oligomers consist of two isoforms, α X-A and α X-B which share high sequence similarity and define the common α -Crystallin fold found in many small heat shock proteins (sHSPs). α X-A and α X-B are hypothesized to play two important roles within the lens. First, α X-A and α X-B belong to a group of proteins called Crystallins (α , β , and γ ) that are very stable proteins that play a role in preserving a uniform density within the lens, which allows it to focus light. The Crystallin proteins’ ability to form diverse and stable oligomers results in a glass-like rather than crystalline organization to the lens protein material, which also aids in the long-term stability of this high-density protein organ. Second, α X-A and α X-B both function as sHSPs that bind to misfolded proteins, preventing formation of large, insoluble protein aggregates (the beginning of cataracts). Our lab is investigating the molecular interactions between α X-A and α X-B that result in its stability, diverse oligomerization, and chaperone function. To this end we are using a model, inducible misfolding protein (insulin B-chain) to study chaperone function by light scatter under various conditions. We are also using random and targeted modification of α X-A and α X-B to simulate long-term protein damage and degradation observed in aged lenses. Focus on the C-terminal strand exchange observed in recent crystal structures and proposed to aid in α X-A and α X-B polydisperse oligomerization is additionally aiding experimental design. We hope to identify specific molecular interactions that result in α X-A and α X-B’s chaperone function, and determine how those interactions relate to stability and self-oligomerization.