SUMMARY
Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that were implicated in the pathology of diabetes, chronic inflammation, Alzheimer’s disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). Nε-carboxy-methyl-lysine (CML) and Nε-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL) modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.
Diabetes-induced
hyperglycemia increases the extracellular concentration
of methylglyoxal. Methylglyoxal-derived hydroimidazolones (MG-H) form
advanced glycation end products (AGEs) that accumulate in the serum
of diabetic patients. The binding of hydroimidozolones to the receptor
for AGEs (RAGE) results in long-term complications of diabetes typified
by vascular and neuronal injury. Here we show that binding of methylglyoxal-modified
albumin to RAGE results in signal transduction. Chemically synthesized
peptides containing hydroimidozolones bind specifically to the V domain
of RAGE with nanomolar affinity. The solution structure of an MG-H1–V
domain complex revealed that the hydroimidazolone moiety forms multiple
contacts with a positively charged surface on the V domain. The high
affinity and specificity of hydroimidozolones binding to the V domain
of RAGE suggest that they are the primary AGE structures that give
rise to AGEs–RAGE pathologies.
Ion mobility spectrometry (IMS), and particularly differential or field asymmetric waveform IMS (FAIMS), was recently shown capable of separating post-translationally modified peptides with variant PTM localization. However, that work was limited to a model peptide with Ser phosphorylation on fairly distant alternative sites. Here, we demonstrate that FAIMS (coupled to ESI/MS) can broadly baseline-resolve variant phosphopeptides from a biologically modified human protein, including those involving phosphorylation of different residues and adjacent sites that challenge existing MS/MS methods most. Singly and doubly phosphorylated variants can be resolved equally well and identified without dissociation, based on accurate separation properties. The spectra change little over a range of infusion solvent pH, hence the present approach should be viable in conjunction with chromatographic separations using mobile phase gradients.
Repeat
length disease thresholds vary among the 10 expanded polyglutamine
(polyQ) repeat diseases, from about 20 to about 50 glutamine residues.
The unique amino acid sequences flanking the polyQ segment are thought
to contribute to these repeat length thresholds. The specific portions
of the flanking sequences that modulate polyQ properties are not always
clear, however. This ambiguity may be important in Huntington’s
disease (HD), for example, where in vitro studies
of aggregation mechanisms have led to distinctly different mechanistic
models. Most in vitro studies of the aggregation
of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism
have been conducted on inexact molecules that are imprecise either
on the N-terminus (recombinantly produced peptides) or on the C-terminus
(chemically synthesized peptides). In this paper, we investigate the
aggregation properties of chemically synthesized HTT exon1 peptides
that are full-length and complete, containing both normal and expanded
polyQ repeat lengths, and compare the results directly to previously
investigated molecules containing truncated C-termini. The results
on the full-length peptides are consistent with a two-step aggregation
mechanism originally developed based on studies of the C-terminally
truncated analogues. Thus, we observe relatively rapid formation of
spherical oligomers containing from 100 to 600 HTT exon1 molecules
and intermediate formation of short protofibril-like structures containing
from 500 to 2600 molecules. In contrast to this relatively rapid assembly,
mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal
HTT exon1 aggregates to disappear in vivo after HTT
production is discontinued.
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