Rama Kannan

The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1-S phase transition and reducing apoptosis.

Trefoil factor (TFF)1 is synthesized and secreted by the normal stomach mucosa and by the gastrointestinal cells of injured tissues. The link between mouse TFF1 inactivation and the fully penetrant antropyloric tumor phenotype prompted the classification of TFF1 as a gastric tumor suppressor gene. Accordingly, altered expression, deletion, and/or mutations of the TFF1 gene are frequently observed in human gastric carcinomas. The present study was undertaken to address the nature of the cellular and molecular mechanisms targeted by TFF1 signalling. TFF1 effects were investigated in IEC18, HCT116, and AGS gastrointestinal cells treated with recombinant human TFF1, and in stably transfected HCT116 cells synthesizing constitutive or doxycycline-induced human TFF1. We observed that TFF1 triggers two types of cellular responses. On one hand, TFF1 lowers cell proliferation by delaying G1-S cell phase transition. This results from a TFF1-mediated increase in the levels of cyclin-dependent kinase inhibitors of both the INK4 and CIP subfamilies, leading to lower E2F transcriptional activity. On the other hand, TFF1 protects cells from chemical-, anchorage-free–, or Bad-induced apoptosis. In this process, TFF1 signalling targets the active form of caspase-9. Together, these results provide the first evidence of a dual antiproliferative and antiapoptotic role for TFF1. Similar paradoxical functions have been reported for tumor suppressor genes involved in cell differentiation, a function consistent with TFF1.

Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker–BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.

The critical role of the central hydrophobic core (residues 71–77) of amyloid-forming αA66-80 peptide in α-crystallin aggregation: a systematic proline replacement study

Abstract Age-related cataract formation is marked by the progressive aggregation of lens proteins. The formation of protein aggregates in the aging lens has been shown to correlate with the progressive accumulation of a range of post-translational crystallin modifications, including oxidation, deamidation, racemization, methylation, acetylation, N- and C-terminal truncations and low molecular weight (LMW) crystallin fragments. We found that an αA-crystallin-derived peptide, αA66-80 (1.8 kDa), is a prominent LMW peptide concentrated in water-insoluble fractions of the aging lens. The peptide has amyloid-like properties and preferentially insolubilizes α-crystallin from lens-soluble fractions. It binds at multiple sites and forms a hydrophobically driven non-covalent complex with α-crystallin to induce α-crystallin aggregation. To define the specific role of the αA66-80 peptide in age-related protein aggregation and cataract formation, it is important to understand the mechanisms by which this peptide acts. We used scanning proline mutagenesis to identify which particular sequences of the peptide drive it to form amyloid-like fibrils and induce α-crystallin aggregation. The secondary structure and the aggregate morphology of the peptides were determined using circular dichroism and transmission electron microscopy, respectively. Peptides were also tested for their ability to induce α-crystallin aggregation. We found that proline replacement of any residue in the sequence FVIFLDV, which corresponds to residues 71–77, led to an absence of both fibril formation and α-crystallin aggregation. The apparently critical role of 71–77 residues in αA66-80 explains their significance in the self-assembly processes of the peptide and further provide insights into the mechanism of peptide-induced aggregation. Our findings may have applications in the design of peptide aggregation inhibitors.

Proteases in Lens and Cataract

The eye lens is a transparent tissue with a unique cellular architecture and pattern of development. Several proteases play pivotal roles during lens morphogenesis and thereafter to maintain the lens transparency. However, with aging, the activity of a number of lens proteases changes, resulting in the degradation of lens proteins and the accumulation of protein fragments. Recent studies suggest that some of the protein fragments that accumulate in the lens with age might be involved in aggregation of lens proteins and development of opacity, also known as cataract. In vitro and in vivo experiments show that lens opacity from α-crystallin aggregation bears the hallmarks of increased proteolysis and decreased α-crystallin chaperone activity. A number of lens proteases are now implicated in the degradation of cytoskeletal proteins, the truncation of lens crystallins, and the generation of peptides that have a role in aggregation and precipitation of lens proteins culminating in cataract. While a perfect animal model to understand the mechanisms of cataractogenesis has yet to be developed, the experimental models now available, such as transgenic mice that overexpress acylpeptide hydrolase, allow study of the lens proteases and the potential therapeutic targets.