Proteins protein cross-links

Several mechanisms have been proposed to explain HIAR the cleavage of protein-protein cross-links,3,6 the disruption of cross-links involving calcium ions,5 an increase in tissue permeability for antibodies, and the removal of trace amounts of paraffin in the tissues. However, recent studies have indicated that the fundamental mechanism of HIAR is based on the cleavage of protein-protein cross-links. 

Hall, D.B., and Struhl, K. (2002) The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo.]. Biol. Chem. 277, 46043-46050. 

Although camosine s ability to suppress both formation and reactivity of some of the age-associated macromolecular modifications (e.g., protein-protein cross-links and protein AGEs) could contribute to its apparent antisenescent effects, these prophylactic actions cannot by themselves explain the dipeptide s apparent rejuvenating activity towards cultured human fibroblasts observed by McFarland and Holliday (1994,... 

Several lines of evidence demonstrate that the active unit of integrase is a multimer. It is clear, as an isolated protein in solution, that integrase forms dimers [6,10-12], and it has been shown by sedimentation equilibrium studies that Rous sarcoma virus (RS V) integrase exists in reversible equilibrium between monomeric, dimeric, and tetrameric forms [13]. Protein-protein cross-linking studies of HIV-1 [14] and RSV [15] integrases confirm the existence of protein dimers and tetramers in solution, and in vivo, the yeast GAL4 two-hybrid system has demonstrated that HIV-1 integrase can interact with itself [16]. 

The protocol for using GMBS or sulfo-GMBS in protein-protein cross-linking applications is similar to that of SMCC or sulfo-SMCC (see Section 1.3). 

Lipid peroxidation is one of the major sources of free-radical mediated injury that directly damages membranes and generates a number of secondary products. In particular, markers of lipid peroxidation have been found to be elevated in brain tissues and body fluids in several neurodegenerative diseases, and the role of lipid peroxidation has been extensively discussed in the context of their pathogenesis. Peroxidation of membrane lipids can have numerous effects, including increased membrane rigidity, decreased activity of membrane-bound enzymes (e.g., sodium pumps), altered activity of membrane receptors, and altered permeability [Anzai et al., 1999 Yehuda et al., 2002], In addition to effects on phospholipids, lipid-initiated radicals can also directly attack membrane proteins and induce lipid-lipid, lipid-protein, and protein-protein cross-linking, all of which obviously have effects on membrane function. 

The key to hexavalent chromium s mutagenicity and possible carcinogenicity is the abiHty of this oxidation state to penetrate the cell membrane. The Cr(VI) Species promotes DNA strand breaks and initiates DNA—DNA and DNA-protein cross-links both in cell cultures and in vivo (105,112,128—130). The mechanism of this genotoxic interaction may be the intercellular reduction of Cr(VI) in close proximity to the nuclear membrane. When in vitro reductions of hexavalent chromium are performed by glutathione, the formation of Cr(V) and glutathione thiyl radicals are observed, and these are beHeved to be responsible for the formation of the DNA cross-links (112). 

Proteins that cross-link actin filaments bind to their sides to produce bundles or three-dimensional networks (Otto, 1994). In microvilli, approximately 20 actin filaments of the core are cross-linked by villin (95 kD) and fimbrin (68 kD) in helical array to form a compact bundle (Figure 5). Filamin (2 x 250 kD) induces the formation of an actin network with gel formation. By immunofluorescence microscopy, this ABP is found in the ruffled, motile edge of cultured cells, where only actin filaments are abundant. 

Chromosomal aberrations Gene mutation Dominant lethal mutation Micronucleus formation Micronucleus formation Micronucleus formation Chromosomal aberrations Sister chromatid exchange Micronucleus formation Chromosomal aberrations Sister chromatid exchange DNA-protein cross-links Nondisjunction of Y chromosome in sperm DNA damage (single-strand breaks)... 

Brownlee, M., Vlassara, H., Kooney, A., Ulrich, P. and Cerami, A. (1986). Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232, 1629-1632. 

Nackerdien, Z., Rao, G., Cacciuttolo, M.A., Gajewski, E. and Dizdaroglu, M. (1991). Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions. Biochemistry 30, 4872-4879. 

Oxo-G (81), spiroaminodihydantoin (83), and guanidinohydantoin (85) are mutagenic lesions in duplex DNA. ° Interestingly, the electrophilic intermediate generated upon oxidation 8-oxo-G can yield protein-DNA cross-links. ... 

Interestingly, the nucleophilic addition of water in the sequence of events giving rise to 41 represents a relevant model system for investigating the mechanism of the generation of DNA-protein cross-links under radical-mediated oxidative conditions [80, 81]. Thus, it was shown that lysine tethered to dGuo via the 5 -hydroxyl group is able to participate in an intramolecular cyclization reaction with the purine base at C-8, subsequent to one electron oxidation [81]. 

Although formaldehyde penetrates very quickly, its protein cross-linking fixative effects are not as immediate. Modifications to this and improvements on the model used originally in studies by Baker et al.,7 Fox et al.,8 and Helander9 have demonstrated that in tissue and in these more complex models, the penetration and therefore Medawar s constant for formaldehyde is not as great as 5.5 and is probably more like 3.6.7... 

Finally temperature and tissue type do have an effect on the penetration and fixation. This, combined with a failure to appreciate protein cross-linking time or actual fixation time required for formaldehyde, is probably the main reason for intra- and interlaboratory variation. 

During the tissue fixation process, proteins are cross-linked, causing some epitopes to become undetectable by the staining protocols.10 HIAR reverses this effect, allowing these epitopes to be stained, and therefore has become increasingly important for many IHC staining protocols.19-22 However, the available automated IHC platforms vary in their ability to perform online HIAR. 

Fraenkel-Conrat H, Olcott HS. Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 1948 174 827-843. 

Fraenkel-Conrat H, Olcott HS. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 1948 70 2673-2684. 

A second type of protein cross-link has been proposed to occur between a secondary amine and a carbonyl compound through the Mannich reaction20... 

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

---