Principal Investigator/Program Director (Last, first, middle): Mchaourab, Hassane, S. RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? m Yes l No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes m No IACUC Approval Date: Animal Welfare Assurance Number 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 2725-projsumm.pdf Mime Type: application/pdf 7. * Project Narrative 4928-narrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 3192-biblio.pdf Mime Type: application/pdf 9. Facilities &Other Resources 8937-RESOURCES.pdf Mime Type: application/pdf 10. Equipment 8390-SummaryEQUIPMENT.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Mchaourab, Hassane, S. Project Summary The """"""""wear and tear"""""""" of lens aging is recorded at the molecular level through accumulated modifications of the crystallins in its fiber cells. This slow environmental perturbation in the absence of protein turn-over leads to a progressive loss in crystallins stability and solubility and alters protein-protein interactions, all of which compromise lens transparency and refractivity. Roughly 35% of the fiber cell weight consists of the molecular chaperone, ?-crystallin, a protein-stability sensor that binds aggregation-prone proteins. Attractive interactions between ?-crystallin and destabilized ?- and ?-crystallins are a central facet of lens aging. The exhaustion of chaperone capacity is hypothesized to be a central catalyst for age-related cataract. Furthermore, the earliest stage of age-related nuclear cataract is temporally correlated with the appearance of disulfide cross-linked aggregates of crystallins. The long term goal of this grant is to develop an understanding of the interrelationships between crystallins stability, chaperone structure, affinity and capacity, and molecular crowding in the process of lens aging and the development of cataract. In the next funding period, we will test two hypotheses regarding the molecular mechanisms of age-related and hereditary cataracts.
Aims 1 and 2 will undertake a systematic analysis of the energetic threshold required to trigger binding of destabilized ?- and ?-crystallins to ?-crystallin and determine whether chaperone-driven interactions lead to formation of disulfide cross-links through the entropic advantage afforded by complex formation. Critical to this endeavor is the use of an innovative label-free approach, backscattering interferometry, which allows characterization of these interactions in real time. We will also test the hypothesis that ?A-crystallin cysteine mutants linked to congenital cataract lead to a compressed aging process from the perspective of titration of ?A-crystallin capacity and formation of aggregates. Based on preliminary data, complex formation by these mutants is driven by their increased affinity and capacity rather than by substrate destabilization and results in the formation of disulfide cross-links.
In aim 3, state of the art structural tools will be employed to provide snapshots of a chaperone activated state and its complex with the substrate. These studies will complement the mechanistic insight of aims 1 and 2 by identifying sequence and structural motifs involved in binding, and defining the basis of activation by oligomer expansion of small heat-shock proteins. Project Description Page 6

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BDCN-F (02))
Program Officer
Araj, Houmam H
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Mishra, Sanjay; Chandler, Shane A; Williams, Dewight et al. (2018) Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity. Structure 26:1116-1126.e4
Mishra, Sanjay; Wu, Shu-Yu; Fuller, Alexandra W et al. (2018) Loss of ?B-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart. J Biol Chem 293:740-753
Wu, Shu-Yu; Zou, Ping; Fuller, Alexandra W et al. (2016) Expression of Cataract-linked ?-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability. J Biol Chem 291:25387-25397
Koteiche, Hanane A; Claxton, Derek P; Mishra, Sanjay et al. (2015) Species-Specific Structural and Functional Divergence of ?-Crystallins: Zebrafish ?Ba- and Rodent ?A(ins)-Crystallin Encode Activated Chaperones. Biochemistry 54:5949-58
Anderson, David M G; Floyd, Kyle A; Barnes, Stephen et al. (2015) A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging. Anal Bioanal Chem 407:2311-20
Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A et al. (2015) A conserved role of ?A-crystallin in the development of the zebrafish embryonic lens. Exp Eye Res 138:104-13
Shi, Jian; Koteiche, Hanane A; McDonald, Ezelle T et al. (2013) Cryoelectron microscopy analysis of small heat shock protein 16.5 (Hsp16.5) complexes with T4 lysozyme reveals the structural basis of multimode binding. J Biol Chem 288:4819-30
Yirdaw, Robel B; McHaourab, Hassane S (2012) Direct observation of T4 lysozyme hinge-bending motion by fluorescence correlation spectroscopy. Biophys J 103:1525-36
Mishra, Sanjay; Stein, Richard A; McHaourab, Hassane S (2012) Cataract-linked ýýD-crystallin mutants have weak affinity to lens chaperones ýý-crystallins. FEBS Lett 586:330-6
McHaourab, Hassane S; Lin, Yi-Lun; Spiller, Benjamin W (2012) Crystal structure of an activated variant of small heat shock protein Hsp16.5. Biochemistry 51:5105-12

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