protein aggregation
{{Short description|Accumulation of clumps of misfolded or disordered proteins}}
File:Protein Aggregation.jpgs, get degraded, or refold back to its native structure.]]
In molecular biology, protein aggregation is a phenomenon in which intrinsically-disordered or mis-folded proteins aggregate (i.e., accumulate and clump together) either intra- or extracellularly.{{cite journal | vauthors = Aguzzi A, O'Connor T | title = Protein aggregation diseases: pathogenicity and therapeutic perspectives | journal = Nature Reviews. Drug Discovery | volume = 9 | issue = 3 | pages = 237–248 | date = March 2010 | pmid = 20190788 | doi = 10.1038/nrd3050 | s2cid = 5756683 }}{{cite journal | vauthors = Stefani M, Dobson CM | title = Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution | journal = Journal of Molecular Medicine | volume = 81 | issue = 11 | pages = 678–699 | date = November 2003 | pmid = 12942175 | doi = 10.1007/s00109-003-0464-5 | s2cid = 23544974 }} Protein aggregates have been implicated in a wide variety of diseases known as amyloidoses, including ALS, Alzheimer's, Parkinson's and prion disease.{{cite journal | vauthors = De Felice FG, Vieira MN, Meirelles MN, Morozova-Roche LA, Dobson CM, Ferreira ST | title = Formation of amyloid aggregates from human lysozyme and its disease-associated variants using hydrostatic pressure | journal = FASEB Journal | volume = 18 | issue = 10 | pages = 1099–1101 | date = July 2004 | pmid = 15155566 | doi = 10.1096/fj.03-1072fje | doi-access = free | s2cid = 13647147 }}{{cite journal | vauthors = Tanzi RE, Bertram L | title = Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective | journal = Cell | volume = 120 | issue = 4 | pages = 545–555 | date = February 2005 | pmid = 15734686 | doi = 10.1016/j.cell.2005.02.008 | s2cid = 206559875 | doi-access = free }}
After synthesis, proteins typically fold into a particular three-dimensional conformation that is the most thermodynamically favorable: their native state.{{cite journal | vauthors = Brüning A, Jückstock J | title = Misfolded proteins: from little villains to little helpers in the fight against cancer | journal = Frontiers in Oncology | volume = 5 | pages = 47 | date = 2015-01-01 | pmid = 25759792 | pmc = 4338749 | doi = 10.3389/fonc.2015.00047 | doi-access = free }} This folding process is driven by the hydrophobic effect: a tendency for hydrophobic (water-fearing) portions of the protein to shield themselves from the hydrophilic (water-loving) environment of the cell by burying into the interior of the protein. Thus, the exterior of a protein is typically hydrophilic, whereas the interior is typically hydrophobic.
Protein structures are stabilized by non-covalent interactions and disulfide bonds between two cysteine residues. The non-covalent interactions include ionic interactions and weak van der Waals interactions. Ionic interactions form between an anion and a cation and form salt bridges that help stabilize the protein. Van der Waals interactions include nonpolar interactions (i.e. London dispersion force) and polar interactions (i.e. hydrogen bonds, dipole-dipole bond). These play an important role in a protein's secondary structure, such as forming an alpha helix or a beta sheet, and tertiary structure. Interactions between amino acid residues in a specific protein are very important in that protein's final structure.
When there are changes in the non-covalent interactions, as may happen with a change in the amino acid sequence, the protein is susceptible to misfolding or unfolding. In these cases, if the cell does not assist the protein in re-folding, or degrade the unfolded protein, the unfolded/misfolded protein may aggregate, in which the exposed hydrophobic portions of the protein may interact with the exposed hydrophobic patches of other proteins.{{cite journal | vauthors = Gething MJ, Sambrook J | title = Protein folding in the cell | journal = Nature | volume = 355 | issue = 6355 | pages = 33–45 | date = January 1992 | pmid = 1731198 | doi = 10.1038/355033a0 | s2cid = 4330003 | bibcode = 1992Natur.355...33G }}{{cite journal | vauthors = Roberts CJ | title = Non-native protein aggregation kinetics | journal = Biotechnology and Bioengineering | volume = 98 | issue = 5 | pages = 927–938 | date = December 2007 | pmid = 17705294 | doi = 10.1002/bit.21627 | s2cid = 21787377 | doi-access = free }} There are three main types of protein aggregates that may form: amorphous aggregates, oligomers, and amyloid fibrils.{{Cite book|title=Lehninger Principles of Biochemistry| vauthors = Cox DL, Nelson MM |publisher=W.H. Freeman|year=2013|isbn=978-1-4292-3414-6|location=New York|pages=143}}
Causes
Protein aggregation can occur due to a variety of causes. There are four classes that these causes can be categorized into, which are detailed below.
= Mutations =
Mutations that occur in the DNA sequence may or may not affect the amino acid sequence of the protein. When the sequence is affected, a different amino acid may change the interactions between the side chains that affect the folding of the protein. This can lead to exposed hydrophobic regions of the protein that aggregate with the same misfolded/unfolded protein or a different protein.{{cite journal | vauthors = Palanikumar L, Karpauskaite L, Al-Sayegh M, Chehade I, Alam M, Hassan S, Maity D, Ali L, Kalmouni M, Hunashal Y, Ahmed J, Houhou T, Karapetyan S, Falls Z, Samudrala R, Pasricha R, Esposito G, Afzal AJ, Hamilton AD, Kumar S, Magzoub M | display-authors = 6 | title = Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function | journal = Nature Communications | volume = 12 | issue = 1 | pages = 3962 | date = June 2021 | pmid = 34172723 | pmc = 8233319 | doi = 10.1038/s41467-021-23985-1 | bibcode = 2021NatCo..12.3962P }}
In addition to mutations in the affected proteins themselves, protein aggregation could also be caused indirectly through mutations in proteins in regulatory pathways such as the refolding pathway (molecular chaperones) or the ubiquitin-proteasome pathway (ubiquitin ligases).{{cite journal | vauthors = Berke SJ, Paulson HL | title = Protein aggregation and the ubiquitin proteasome pathway: gaining the UPPer hand on neurodegeneration | journal = Current Opinion in Genetics & Development | volume = 13 | issue = 3 | pages = 253–261 | date = June 2003 | pmid = 12787787 | doi = 10.1016/S0959-437X(03)00053-4 }} Chaperones help with protein refolding by providing a safe environment for the protein to fold. Ubiquitin ligases target proteins for degradation through ubiquitin modification.{{cite book | vauthors = Grillari J, Grillari-Voglauer R, Jansen-Dürr P |title=Post-translational modification of cellular proteins by ubiquitin and ubiquitin-like molecules: role in cellular senescence and aging | veditors = Urbani F, Magariños R, Puertas S, Carretero M, Rodriguez V, Liñares R, Bravo JM, Lapuente R, Seoane FJ, Iglesias V | display-editors = 6 |year=2010 |pages=172–196 |publisher=Springer Science+Business Media |isbn=978-1-4419-7002-2 | name-list-style = vanc }}
= Problems with protein synthesis =
Protein aggregation can be caused by problems that occur during transcription or translation. During transcription, DNA is copied into mRNA, forming a strand of pre-mRNA that undergoes RNA processing to form mRNA.{{Cite book|title=Molecular Biology| vauthors = Weaver RF |publisher=McGraw-Hill|year=2012|isbn=978-0-07-352532-7|location=New York|pages=122–156, 523–600}} During translation, ribosomes and tRNA help translate the mRNA sequence into an amino acid sequence. If problems arise during either step, making an incorrect mRNA strand and/or an incorrect amino acid sequence, this can cause the protein to misfold, leading to protein aggregation.{{cn|date=June 2022}}
= Environmental stresses =
Environmental stresses such as extreme temperatures and pH or oxidative stress can also lead to protein aggregation.{{cite journal | vauthors = Tyedmers J, Mogk A, Bukau B | title = Cellular strategies for controlling protein aggregation | journal = Nature Reviews. Molecular Cell Biology | volume = 11 | issue = 11 | pages = 777–788 | date = November 2010 | pmid = 20944667 | doi = 10.1038/nrm2993 | s2cid = 22449895 }} One such disease is cryoglobulinemia.
Extreme temperatures can weaken and destabilize the non-covalent interactions between the amino acid residues. pHs outside of the protein's pH range can change the protonation state of the amino acids, which can increase or decrease the non-covalent interactions. This can also lead to less stable interactions and result in protein unfolding.
Oxidative stress can be caused by radicals such as reactive oxygen species (ROS). These unstable radicals can attack the amino acid residues, leading to oxidation of side chains (e.g. aromatic side chains, methionine side chains) and/or cleavage of the polypeptide bonds.{{cite journal | vauthors = Stadtman ER, Levine RL | title = Free radical-mediated oxidation of free amino acids and amino acid residues in proteins | journal = Amino Acids | volume = 25 | issue = 3–4 | pages = 207–218 | date = December 2003 | pmid = 14661084 | doi = 10.1007/s00726-003-0011-2 | s2cid = 26844881 }} This can affect the non-covalent interactions that hold the protein together correctly, which can cause protein destabilization, and may cause the protein to unfold.
= Aging =
Cells have mechanisms that can refold or degrade protein aggregates. However, as cells age, these control mechanisms are weakened and the cell is less able to resolve the aggregates.
The hypothesis that protein aggregation is a causative process in aging is testable now since some models of delayed aging are in hand. If the development of protein aggregates was an aging independent process, slowing down aging will show no
effect on the rate of proteotoxicity over time. However, if aging is associated
with decline in the activity of protective mechanisms against proteotoxicity,
the slow aging models would show reduced aggregation and proteotoxicity. To
address this problem several toxicity assays have been done in C. elegans.
These studies indicated that reducing the activity of insulin/IGF signaling
(IIS), a prominent aging regulatory pathway protects from
neurodegeneration-linked toxic protein aggregation. The validity of this approach
has been tested and confirmed in mammals as reducing the activity of the IGF-1
signaling pathway protected Alzheimer's model mice from the behavioral and
Aggregate localization
Several studies have shown that cellular responses to protein aggregation are well-regulated and organized. Protein aggregates localize to specific areas in the cell, and research has been done on these localizations in prokaryotes (E.coli) and eukaryotes (yeast, mammalian cells).{{cite journal | vauthors = Coquel AS, Jacob JP, Primet M, Demarez A, Dimiccoli M, Julou T, Moisan L, Lindner AB, Berry H | display-authors = 6 | title = Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect | journal = PLOS Computational Biology | volume = 9 | issue = 4 | pages = e1003038 | date = April 2013 | pmid = 23633942 | doi = 10.1371/journal.pcbi.1003038 | pmc = 3636022 | arxiv = 1303.1904 | bibcode = 2013PLSCB...9E3038C | doi-access = free }} From the macroscopic point of view, positron emission tomography tracers are used for certain misfolded proitein.{{cite journal | vauthors = Scott JJ | title = The reinnervation of cat muscle spindles by skeletofusimotor axons | journal = Brain Research | volume = 401 | issue = 1 | pages = 152–154 | date = January 1987 | pmid = 2949798 | doi = 10.1016/0006-8993(87)91175-9 | s2cid = 42447184 }} Recently, a team of researchers led by Dr. Alessandro Crimi has proposed a machine learning method to predict future deposition in the brain. {{cite journal | vauthors = Gherardini L, Zajdel A, Pini L, Crimi A | title = Prediction of misfolded proteins spreading in Alzheimer's disease using machine learning and spreading models | journal = Cerebral Cortex | date = October 2023 | volume = 33 | issue = 24 | pages = 11471–11485 | pmid = 37833822 | doi = 10.1093/cercor/bhad380 | doi-access = free | pmc = 10724880 }}
= Bacteria =
The aggregates in bacteria asymmetrically end up at one of the poles of the cell, the "older pole." After the cell divides, the daughter cells with the older pole gets the protein aggregate and grows more slowly than daughter cells without the aggregate. This provides a natural selection mechanism for reducing protein aggregates in the bacterial population.{{cite journal | vauthors = Bednarska NG, Schymkowitz J, Rousseau F, Van Eldere J | title = Protein aggregation in bacteria: the thin boundary between functionality and toxicity | journal = Microbiology | volume = 159 | issue = Pt 9 | pages = 1795–1806 | date = September 2013 | pmid = 23894132 | doi = 10.1099/mic.0.069575-0 | doi-access = free }}
= Yeast =
File:A scheme of a yeast cell harboring JUNQ and IPOD inclusions.png
Most of the protein aggregates in yeast cells get refolded by molecular chaperones. However, some aggregates, such as the oxidatively damaged proteins or the proteins marked for degradation, cannot be refolded. Rather, there are two compartments that they can end up in. Protein aggregates can be localized at the Juxtanuclear quality-control compartment (JUNQ), which is near the nuclear membrane, or at the Insoluble Protein deposit (IPOD), near the vacuole in yeast cells. Protein aggregates localize at JUNQ when they are ubiquitinated and targeted for degradation. The aggregated and insoluble proteins localize at IPOD as a more permanent deposition. There is evidence that the proteins here may be removed by autophagy.{{cite journal | vauthors = Takalo M, Salminen A, Soininen H, Hiltunen M, Haapasalo A | title = Protein aggregation and degradation mechanisms in neurodegenerative diseases | journal = American Journal of Neurodegenerative Disease | volume = 2 | issue = 1 | pages = 1–14 | date = 2013-03-08 | pmid = 23516262 | pmc = 3601466 }} These two pathways work together in that the proteins tend to come to the IPOD when the proteasome pathway is being overworked.
= Mammalian cells =
In mammalian cells, these protein aggregates are termed "aggresomes" and they are formed when the cell is diseased. This is because aggregates tend to form when there are heterologous proteins present in the cell, which can arise when the cell is mutated. Different mutates of the same protein may form aggresomes of different morphologies, ranging from diffuse dispersion of soluble species to large puncta, which in turn bear different pathogenicity.{{cite journal | vauthors = Wan W, Zeng L, Jin W, Chen X, Shen D, Huang Y, Wang M, Bai Y, Lyu H, Dong X, Gao Z, Wang L, Liu X, Liu Y | display-authors = 6 | title = A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells | journal = Angewandte Chemie | volume = 60 | issue = 49 | pages = 25865–25871 | date = December 2021 | pmid = 34562048 | doi = 10.1002/anie.202107943 | s2cid = 237626399 }} The E3 ubiquitin ligase is able to recognize misfolded proteins and ubiquinate them. HDAC6 can then bind to the ubiquitin and the motor protein dynein to bring the marked aggregates to the microtubule organizing center (MTOC). There, they pack together into a sphere that surrounds the MTOC. They bring over chaperones and proteasomes and activate autophagy.{{cite journal | vauthors = Garcia-Mata R, Gao YS, Sztul E | title = Hassles with taking out the garbage: aggravating aggresomes | journal = Traffic | volume = 3 | issue = 6 | pages = 388–396 | date = June 2002 | pmid = 12010457 | doi = 10.1034/j.1600-0854.2002.30602.x | s2cid = 305786 | doi-access = free }}
Elimination
There are two main protein quality control systems in the cell that are responsible for eliminating protein aggregates. Misfolded proteins can get refolded by the bi-chaperone system or degraded by the ubiquitin proteasome system or autophagy.{{cite journal | vauthors = Gregersen N, Bolund L, Bross P | title = Protein misfolding, aggregation, and degradation in disease | journal = Molecular Biotechnology | volume = 31 | issue = 2 | pages = 141–150 | date = October 2005 | pmid = 16170215 | doi = 10.1385/MB:31:2:141 | s2cid = 36403914 }}
= Refolding =
The bi-chaperone system utilizes the Hsp70 (DnaK-DnaJ-GrpE in E. coli and Ssa1-Ydj1/Sis1-Sse1/Fe1 in yeast) and Hsp100 (ClpB in E. coli and Hsp104 in yeast) chaperones for protein disaggregation and refolding.{{cite journal | vauthors = Mogk A, Kummer E, Bukau B | title = Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation | journal = Frontiers in Molecular Biosciences | volume = 2 | pages = 22 | date = 2015-01-01 | pmid = 26042222 | pmc = 4436881 | doi = 10.3389/fmolb.2015.00022 | doi-access = free }}
Hsp70 interacts with the protein aggregates and recruits Hsp100. Hsp70 stabilizes an activated Hsp100. Hsp100 proteins have aromatic pore loops that are used for threading activity to disentangle single polypeptides. This threading activity can be initiated at the N-terminus, C-terminus or in the middle of the polypeptide. The polypeptide gets translocated through Hsp100 in a series of steps, utilizing an ATP at each step. The polypeptide unfolds and is then allowed to refold either by itself or with the help of heat shock proteins.{{cite journal | vauthors = Liberek K, Lewandowska A, Zietkiewicz S | title = Chaperones in control of protein disaggregation | journal = The EMBO Journal | volume = 27 | issue = 2 | pages = 328–335 | date = January 2008 | pmid = 18216875 | pmc = 2234349 | doi = 10.1038/sj.emboj.7601970 }}
= Degradation =
Misfolded proteins can be eliminated through the ubiquitin-proteasome system (UPS). This consists of an E1-E2-E3 pathway that ubiquinates proteins to mark them for degradation. In eukaryotes, the proteins get degraded by the 26S proteasome. In mammalian cells, the E3 ligase, carboxy-terminal Hsp70 interacting protein (CHIP), targets Hsp70-bound proteins. In yeast, the E3 ligases Doa10 and Hrd1 have similar functions on endoplasmic reticulum proteins.{{cite journal | vauthors = Chen B, Retzlaff M, Roos T, Frydman J | title = Cellular strategies of protein quality control | journal = Cold Spring Harbor Perspectives in Biology | volume = 3 | issue = 8 | pages = a004374 | date = August 2011 | pmid = 21746797 | pmc = 3140689 | doi = 10.1101/cshperspect.a004374 }} On the molecular level, degradation rate of aggregates vary from protein to protein due to their different internal environments, and thus different accessibility for protease molecules.{{cite journal | vauthors = Wan W, Zeng L, Jin W, Chen X, Shen D, Huang Y, Wang M, Bai Y, Lyu H, Dong X, Gao Z, Wang L, Liu X, Liu Y | display-authors = 6 | title = A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells | journal = Angewandte Chemie | volume = 60 | issue = 49 | pages = 25865–25871 | date = December 2021 | pmid = 34562048 | doi = 10.1002/anie.202107943 | s2cid = 237626399 }}
Misfolded proteins can also be eliminated through autophagy, in which the protein aggregates are delivered to the lysosome.
Toxicity
Although it has been thought that the mature protein aggregates themselves are toxic, evidence suggests that it is in fact immature protein aggregates that are most toxic.{{cite journal | vauthors = Zhu YJ, Lin H, Lal R | title = Fresh and nonfibrillar amyloid beta protein(1-40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AbetaP-channel-mediated cellular toxicity | journal = FASEB Journal | volume = 14 | issue = 9 | pages = 1244–1254 | date = June 2000 | pmid = 10834946 | doi = 10.1096/fasebj.14.9.1244 | doi-access = free | s2cid = 42263619 }}{{cite journal | vauthors = Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Näslund J, Lannfelt L | display-authors = 6 | title = The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation | journal = Nature Neuroscience | volume = 4 | issue = 9 | pages = 887–893 | date = September 2001 | pmid = 11528419 | doi = 10.1038/nn0901-887 | s2cid = 13516479 }} The hydrophobic patches of these aggregates can interact with other components of the cell and damage them. The hypotheses are that the toxicity of protein aggregates is related to mechanisms of the sequestration of cellular components, the generation of reactive oxygen species and the binding to specific receptors in the membrane or through the disruption of membranes.{{cite journal | vauthors = Soto C | title = Unfolding the role of protein misfolding in neurodegenerative diseases | journal = Nature Reviews. Neuroscience | volume = 4 | issue = 1 | pages = 49–60 | date = January 2003 | pmid = 12511861 | doi = 10.1038/nrn1007 | s2cid = 205499427 }} A quantitative assay has been used to determine that higher molecular weight species are responsible for the membrane permeation.{{cite journal | vauthors = Flagmeier P, De S, Wirthensohn DC, Lee SF, Vincke C, Muyldermans S, Knowles TP, Gandhi S, Dobson CM, Klenerman D | display-authors = 6 | title = Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates | journal = Angewandte Chemie | volume = 56 | issue = 27 | pages = 7750–7754 | date = June 2017 | pmid = 28474754 | pmc = 5615231 | doi = 10.1002/anie.201700966 }} It is known that protein aggregates in vitro can destabilize artificial phospholipid bilayers, leading to permeabilization of the membrane.{{cn|date=June 2022}}
In biomanufacturing
Protein aggregation is also a common phenomenon in the biopharmaceutical manufacturing process, which may pose risks to patients via generating adverse immune responses.{{cite journal | vauthors = Vázquez-Rey M, Lang DA | title = Aggregates in monoclonal antibody manufacturing processes | journal = Biotechnology and Bioengineering | volume = 108 | issue = 7 | pages = 1494–1508 | date = July 2011 | pmid = 21480193 | doi = 10.1002/bit.23155 | s2cid = 33285577 }}