To determine the effects of deamidation on structural and functional properties of ␣A-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated ␣B-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206 -214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of ␣A mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of ␣-crystallin containing (i) wild type ␣A (WT-␣A): WT-␣B crystallins, (ii) individual ␣A-deamidated mutants:WT-␣B crystallins, and (iii) WT-␣A:individual ␣B-deamidated mutant crystallins. Compared with the WT-␣A, the three ␣A-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroaggregates of WT-␣A and WT-␣B, the heteroaggregates containing deamidated subunits of either ␣A-or ␣B-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-␣A and deamidated ␣B subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated ␣A-and WT-␣B subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of ␣A-crystallin and (b) the presence of WT-␣B in the ␣-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.␣-Crystallin, the most abundant protein in lens mature fiber cells, constitutes ϳ35% of the total lens protein. In vivo, the ␣A and ␣B subunits at a ratio of 3:1 form an oligomer of 800 kDa. Both ␣A-and ␣B-crystallins are small heat shock proteins (Hsps), 1 and show molecular chaperone activity to protect proteins from aggregation in the eye lens (1, 2). Because of this property, ␣-crystallin is believed to play a crucial role in maintaining the lens transparency. Like other small heat shock proteins, ␣-crystallin also contains a highly conserved sequence of 80 -100 residues (residues 62-143 in ␣A-and 66 -147 in ␣B-crystallin) called the ␣-crystallin domain (3, 4). Based on similarities with the structure of other Hsps, it is believed that the N-terminal region (residues 1-62 in ␣A-and 1-66 in ␣B-crystallin(s)) of ␣-crystallin forms an independently folded domain, whereas the C terminus (referred as the C-terminal extension; residues 143-173 in ␣A-and 147-175 in ␣B-crystallin) is flexible and unstructured (4). Previous reports show that the removal of N-terminal residues (56 residues) and C-terminal extensions (32-34 residues) of ␣A-and ␣B-crystallins lead to improper folding,...

We characterized the mutational landscape of human skin cutaneous melanoma (SKCM) using data obtained from The Cancer Genome Atlas (TCGA) project. We analyzed next-generation sequencing data of somatic copy number alterations and somatic mutations in 303 metastatic melanomas. We were able to confirm preeminent drivers of melanoma as well as identify new melanoma genes. The TCGA SKCM study confirmed a dominance of somatic BRAF mutations in 50% of patients. The mutational burden of melanoma patients is an order of magnitude higher than of other TCGA cohorts. A multi-step filter enriched somatic mutations while accounting for recurrence, conservation, and basal rate. Thus, this filter can serve as a paradigm for analysis of genome-wide next-generation sequencing data of large cohorts with a high mutational burden. Analysis of TCGA melanoma data using such a multi-step filter discovered novel and statistically significant potential melanoma driver genes. In the context of the Pan-Cancer study we report a detailed analysis of the mutational landscape of BRAF and other drivers across cancer tissues. Integrated analysis of somatic mutations, somatic copy number alterations, low pass copy numbers, and gene expression of the melanogenesis pathway shows coordination of proliferative events by Gs-protein and cyclin signaling at a systems level.

The results show that the deamidation of N146 but not of N78 have profound effects on the structural and functional properties of alphaB-crystallin.

To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human ␣A-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as ␣AN101D-transgene and is considered to be "deamidated" in this study. Mice expressing ␣AN101D-transgene are referred to here CRYAA N101D mice. All of the lines showed the expression of ␣AN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) ␣A-transgene (referred to as CRYAA WT mice), the lenses of CRYAA N101D mice showed (a) altered ␣A-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT ␣A-crystallin, the lenses containing the deamidated ␣A-crystallin also showed an aggregation of ␣A-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated ␣A-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.The ocular lens has a unique cellular architecture consisting of a single layer of cuboidal epithelial cells, which divide and differentiate at the equator into fiber cells (1). The fiber cells elongate, and they synthesize fiber cell-specific proteins, such as AQP0 (aquaporin-0)/MIP (main intrinsic protein), cytoskeletal proteins, and crystallins (1-4). As the new fiber cells are laid down at the lens equator, the older fiber cells are pushed toward the lens core and simultaneously lose their nuclei and organelles while exhibiting very little protein turnover. Among the three classes of the vertebrate lens crystallins (␣-, ␤-, and ␥-crystallins), ␣-crystallins are composed of two primary gene products, A and B, known as ␣A-and ␣B-crystallins, that show ϳ60% amino acid homology and constitute up to 50% of the total lens proteins. Both ␣A-and ␣B-crystallins belong to a family of small heat shock proteins with "chaperone" activity (5, 6). These crystallins are constitutively expressed in both lens epithelial and fiber cells (7) and undergo numerous age-related post-translational modifications (PTMs) 2 that lead to their unfolding, aggregation, and insolubilization. These PTMs eventually lead to accumulation of damaged crystal...

[1] Scale-recursive estimation (SRE) is a Kalman-filter-based methodology, which can be used to produce optimal (in terms of bias and minimum variance) estimates of a field at any desired scale given uncertain and sparse observations at different scales. SRE requires the specification of the state equation, which describes the variability of the precipitation process across scales, and the observation equation, which relates the observations to the state. Typical models for describing the multiscale rainfall variability are the multiplicative cascade models. However, in order to convert them into the additive form needed by SRE, one needs to work in the log space, thus creating a problem in handling zero-intermittency in a satisfactory way. In this paper, we propose an alternative approach, based on a data-driven identification methodology, which operates directly on the data and does not require a prespecified multiscale model structure. Rather, system identification and estimation are performed simultaneously via a likelihood-based expectation-maximization (EM) procedure. The merits of the proposed approach versus approaches based on multiplicative cascade models are explored via several examples of synthetic and real precipitation fields. For practical application the proposed approach will need to be extended to include the temporal evolution of storms. This extension presents theoretical challenges, and until these are addressed, a simple alternative is explored of coupling the EM-SRE approach with a spatial downscaling methodology to merge precipitation observations available at different spatial and temporal scales. An example application is presented motivated by its relevance to the Global Precipitation Measuring (GPM) mission.Citation: Gupta, R., V. Venugopal, and E. Foufoula-Georgiou (2006), A methodology for merging multisensor precipitation estimates based on expectation-maximization and scale-recursive estimation,

Background: We have engaged in an international program designated the Bank On A Cure, which has established DNA banks from multiple cooperative and institutional clinical trials, and a platform for examining the association of genetic variations with disease risk and outcomes in multiple myeloma.

No abstract

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partia...