Proteinaceous
aggregation is a well-known observable in Alzheimer’s
disease (AD), but failure and storage of lysosomal bodies within neurons
is equally ubiquitous and actually precedes bulk accumulation of extracellular
amyloid plaque. In fact, AD shares many similarities with certain
lysosomal storage disorders though establishing a biochemical connection
has proven difficult. Herein, we demonstrate that isomerization and
epimerization, which are spontaneous chemical modifications that occur
in long-lived proteins, prevent digestion by the proteases in the
lysosome (namely, the cathepsins). For example, isomerization of aspartic
acid into
l
-isoAsp prevents digestion of the N-terminal portion
of Aβ by cathepsin L, one of the most aggressive lysosomal proteases.
Similar results were obtained after examination of various target
peptides with a full series of cathepsins, including endo-, amino-,
and carboxy-peptidases. In all cases peptide fragments too long for
transporter recognition or release from the lysosome persisted after
treatment, providing a mechanism for eventual lysosomal storage and
bridging the gap between AD and lysosomal storage disorders. Additional
experiments with microglial cells confirmed that isomerization disrupts
proteolysis in active lysosomes. These results are easily rationalized
in terms of protease active sites, which are engineered to precisely
orient the peptide backbone and cannot accommodate the backbone shift
caused by isoaspartic acid or side chain dislocation resulting from
epimerization. Although Aβ is known to be isomerized and epimerized
in plaques present in AD brains, we further establish that the rates
of modification for aspartic acid in positions 1 and 7 are fast and
could accrue prior to plaque formation. Spontaneous chemistry can
therefore provide modified substrates capable of inducing gradual
lysosomal failure, which may play an important role in the cascade
of events leading to the disrupted proteostasis, amyloid formation,
and tauopathies associated with AD.
Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N-or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photonbased fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15−20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.
Isomerization of
individual residues in long-lived proteins (LLPs)
is a subject of growing interest in connection with many age-related
human diseases. When isomerization occurs in LLPs, it can lead to
deleterious changes in protein structure, function, and proteolytic
degradation. Herein, we present a novel labeling technique for rapid
identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate
formed during reaction of l-isoAsp-containing peptides with
PIMT and S-adenosyl methionine (SAM) is reactive
with Tris base and results in a Tris-modified aspartic acid residue
with a mass shift of +103 Da. Tris-modified aspartic acid exhibits
prominent and repeated neutral loss of water when subjected to collisional
activation. In addition, another dissociation pathway regenerates
the original peptide following loss of a characteristic mass shift.
Furthermore, it is demonstrated that Tris modification can be used
to identify sites of isomerization in LLPs from biological samples
such as the lens of the eye. This approach simplifies identification
by labeling isomerization sites with a tag that causes a mass shift
and provides characteristic loss during collisional activation.
Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin‐dependent leukocyte adhesion or pathogen‐receptor interactions. Mass‐spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose‐containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments—often referred to as fucose migration—has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion‐mobility spectrometry, radical‐directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density‐functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product.
Proteinaceous aggregation is a well-known observable in Alzheimer's disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely the cathepsins). For example, isomerization of aspartic acid into L-isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxypeptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.3
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.