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Spontaneous isomerization of long-lived proteins provides a molecular mechanism for the lysosomal failure observed in Alzheimer’s disease

Tyler R. Lambeth, Dylan L. Riggs, Lance E. Talbert, Jin Tang, Emily Coburn, Amrik S. Kang, Jessica Noll, Catherine Augello, Byron D. Ford, Ryan R. Julian

Posted on: 15 May 2019

Preprint posted on 12 April 2019

Article now published in ACS Central Science at http://dx.doi.org/10.1021/acscentsci.9b00369

Do LSDs have the answer? Exploring a promising link between lysosomal storage disorders and Alzheimer’s disease.

Selected by Joanna Cross

Background

I was drawn to this preprint not by the topic of Alzheimer’s disease (AD), but by the word ‘lysosome’. This may surprise some people, but one of the disorders I studied during my PhD was Niemann-Pick type C disease, a lysosomal storage disorder (LSD) known colloquially as “childhood Alzheimer’s”.  The lysosomal system is the cell’s recycling facility; old proteins are deposited in the lysosomes, and amino acids are regurgitated out, ready to be made into something new and useful.  Within the lysosome, enzymes called endopeptidases cleave proteins at internal sites.  Exopeptidases finish the job by further digesting the resulting smaller peptides from both termini (at this point, I visualize Pac-man style objects consuming the little yellow dots before running head-long into each other).  LSDs arise when enzymes or proteins responsible for the efficient running of the ‘facility’ are faulty: imagine what would happen if the Pac-men lost their mouths.  There would be a lot of un-eaten yellow dots.  Many LSDs occur at a young age, but, like Alzheimer’s disease, are neurogenerative and often fatal.

The focus of this preprint is not on the machinery of the lysosomes, but on the substrates themselves.  In particular, two forms of chemical transformation known as isomerization and epimerization.  Isomerization primarily occurs at aspartic acid, when the side chain inserts into and elongates the peptide backbone (picture what happens when a couple of friends want to join a picture, not at the end, but right in the middle!).  Epimerization is when the side chain of an amino acid changes from the usual L-chirality to the D-chirality: L- and D- forms are mirror images of each other, just like your hands.  So, what is the importance of these two chemical transformations?  As with so many things, nature shows us the answer. Many venoms contain epimerized amino acids to help prevent degradation in the prey’s body, showing that these transformations cause resistance to protease degradation.

Amyloid-b(Ab) and tau are subjected to both chemical transformations and are major constituents of the senile plaques and tangles, respectively, present in AD.  Indeed, isomerized and epimerized Abhas been found in the brains of people with AD.   Therefore, if these chemical changes do indeed prevent lysosomal protein degradation, this could provide the link between LSDs and AD.

 

Key Findings

In order to demonstrate how iso/epi modifications can affect the lysosomal machinery, the authors first subjected synthetic peptides to endopeptidases (cathepsins D and L), and exopeptidases (cathepsins B and H).  The resulting peptide fragments were then measured using Liquid Chromatography Mass Spectrometry (LC-MS).

When a synthetic peptide, derived from aB-crystallin, was exposed to both cathepsins D and L, there was reduced digestion of the iso/epi form compared to the canonical peptide.  As exopeptidases function by breaking down peptides from each termini, a palindromic peptide was synthesized, “RLHTIDITHLR”. Intriguingly, changing the middle asparagine from L-asp to D-isoasp caused persistence of the “IDIT” peptide fragment, even after exposing the peptide to cathepsins B and H for 48 hours. Therefore, these experiments showed that iso/epi modifications can affect digestion by both endo- and exo-peptidases.

But how can iso/epi modifications contribute to the pathology of AD? Abis a peptide comprising 36-43 amino acids and these peptides are the main component of the amyloid plaques present in AD.  The authors used an Abpeptide fragment (Ab1-9) derived from the N-terminal end of this peptide, as this portion contains asparagine residues that are known to be highly isomerized in amyloid plaques.  Similar to the initial experiments, cathepsins B and L were able to significantly digest the canonical peptide but cannot alter the double isomer (L-isoAsp1, D-isoAsp7).   Interestingly, cathepsins D and H were unable to appreciably digest either form of the peptide, suggesting that the N-terminal portion of Abis generally hard to digest. Thus, addition of the iso/epi modifications can further complicate matters.

Given that intracellular aggregates of tau are also a hallmark of AD, the authors also subjected a tau peptide fragment (Tau594IINKKLDL601) to cathepsins.  Tau contains two residues that can be subjected to iso/epi modifications: Asn596and Asp600.  The authors showed that while cathepsins B, L and H could fully digest the canonical sequence, this is prevented by the iso/epi modifications.

Although the four cathepsins used above are the most abundant, they are by no means the only lysosomal proteases present in cells. To explore additional enzymes, the authors exposed mouse microglial cells to a chimeric peptide derived from the N-terminus of Ab.  The peptide included a polyarginine sequence for lysosomal delivery, and a quencher peptide sequence that results in fluorescence when the peptide is cleaved. After 150 minutes, fluorescence was observed for the canonical chimeric peptide, whereas the double isomer produced a significantly lower intensity fluorescence.  This shows that there is not an unknown protease in the lysosomal system that is designed to digest iso/epi sites.  Additionally, these results were recapitulated by incubating the chimeric peptide with only cathepsin L, thereby confirming the validity of the LC-MS approach.

The next question is why these modifications affect their ability to be digested by cathepsins.  To answer this, the authors turned to X-ray crystallography.  By analyzing the crystal structure of a peptide bound to the active site of cathepsin L, it can be seen that several hydrogen bonds orientate the substrate backbone so the cleavage site is accessible. Iso/epi modifications would change the structure so that they cannot fit properly into the active site, thereby preventing cleavage.  This hampered ability of cathepsins to completely digest AD-associated peptides would produce peptide fragments that are too long to be recognized by the transporters responsible for releasing digested amino acids back to the cytosol. This indicates that accumulation of these byproducts in the lysosomes is possible.

 

What I like about this preprint

Since my PhD, I have been intrigued about the link between LSDs and AD, and this preprint puts forward an intriguing and believable option.  Although other proteolytic pathways exist that could also have difficulties dealing with these modifications, lysosomes may be especially vulnerable due to the inability to get rid of undigested byproducts, resulting in failure of the organelle.

Future questions could include:

  • What drives the epi/iso modifications in AD?
  • What mechanisms and pathways are affected by lysosomal failure and how this contributes to the disease pathology?
  • Do drug treatments known to have positive outcomes in LSDs also influence biomarkers of AD, such as the misfolding and aggregation of tau and Ab?

Ultimately, this study represents an intriguing connection between LSDs and AD and provides a novel avenue for study.  If those little Pac-Men could be given back their mouths so they can fully devour all the yellow dots, perhaps we will be a step closer to improving lives for so many people.

 

doi: https://doi.org/10.1242/prelights.10707

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