Transglutaminases (TGases) are defined as enzymes capable of forming isopeptide bonds by transfer of an amine onto glutaminyl residues of a protein. Here we show that the membrane-bound form of the TGase 1 enzyme can also form ester bonds between specific glutaminyl residues of human involucrin and a synthetic analog of epidermal specific -hydroxyceramides. The formation of a Ϸ5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of -hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural -hydroxyceramides N-[16-(16-hydroxyhexadecyl)oxypalmitoyl]-sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles containing lipid Z, lipid Z was attached to involucrin and formed saponifiable protein-lipid adducts. By mass spectroscopy and sequencing of tryptic lipopeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the ␥-carboxamido groups to lipid esters. Several of these residues have been found previously to be attached to ceramides in vivo. Mass spectrometric analysis after acetonide derivatization also revealed that ester formation involved primarily the -hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epidermal barrier formation and provide insights into the pathophysiology of lamellar ichthyosis resulting from defects of TGase 1 enzyme.Terrestrial vertebrates protect themselves from chemical and physical damage and uncontrolled water loss by maintaining a water-impermeable barrier function of their epidermis. In mammals, this function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes termed the cornified envelope (CE) and by impeding water diffusion across the stratum corneum by mortaring the corneocytes together by layers of skin-specific lipids (1, 2). These lipids differ in composition from other bilayerforming lipids found in living cells. Notably, their phospholipid content is lost, and instead they contain increased amounts of free fatty acids, cholesterol and its acyl and sulfate esters, and several classes of ceramides, including epidermal-specific longchain -hydroxy-and -hydroxyacylceramides (3). Synthesis of these lipids is initiated in the spinous layer, and they are temporarily stored in lamellar bodies of stratum granulosum, wherein they are arranged as stacks of tetralaminar sheets. Preceding or paralleling the formation of the protein envelope, the contents of the lamellar bodies are extruded into the intercellular space. One component of these lipids is epidermal-specific long-chain -hydroxyceramides that become covalently attached onto the outer surface of the CE as a Ϸ5-nm monomolecular layer. These protein-linked ceramides interdigitate with the intercellular lipid in a comb-like fashion, presuma...
Transglutaminase (TGase) enzymes catalyze the formation of covalent cross-links between protein-bound glutamines and lysines in a calcium-dependent manner, but the role of Ca 2+ ions remains unclear. The TGase 3 isoform is widely expressed and is important for epithelial barrier formation. It is a zymogen, requiring proteolysis for activity. We have solved the three-dimensional structures of the zymogen and the activated forms at 2.2 and 2.1 A Ê resolution, respectively, and examined the role of Ca 2+ ions. The zymogen binds one ion tightly that cannot be exchanged. Upon proteolysis, the enzyme exothermally acquires two more Ca 2+ ions that activate the enzyme, are exchangeable and are functionally replaceable by other lanthanide trivalent cations. Binding of a Ca 2+ ion at one of these sites opens a channel which exposes the key Trp236 and Trp327 residues that control substrate access to the active site. Together, these biochemical and structural data reveal for the ®rst time in a TGase enzyme that Ca 2+ ions induce structural changes which at least in part dictate activity and, moreover, may confer substrate speci®city.
The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G 1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000-to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.
The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming gamma-glutamyl-epsilon-lysine (GGEL) cross-links between distinct proteins. Here, we show that the frequency of GGEL cross-links is an order of magnitude higher in Alzheimer's brain cortex than in age-matched or younger controls. This difference is due to the accumulation of GGEL cross-links in ubiquitin-immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross-linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to alpha-synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross-linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross-linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross-link two (poly)ubiquitin chains. One lysine residue of parkin and one of alpha-synuclein were also found to be cross-linked. The data suggest that cross-linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.
MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.
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