{{Short description|Types of cytosolic protein inclusion bodies}} {{Redirect|JUNQ|the singer and actor|Jun.Q|the journal publishing null results|Journal of Unsolved Questions}} [[File:A scheme of a yeast cell harboring JUNQ and IPOD inclusions.png|thumb|upright=1.25|Eukaryote cells sort misfolded proteins into two quality control compartments: JUNQ and IPOD, based on their ubiquitination state.]] '''JUNQ and IPOD''' are types of [[cytosolic]] [[protein]] [[inclusion bodies]] in [[eukaryotes]].
[[Neurodegenerative diseases]], such as [[Parkinson's]], [[Alzheimer's]], and [[Huntington's]], are associated and correlated with [[protein aggregation]] and accumulation of [[misfolded proteins]] in [[inclusion bodies]]. For many years, protein aggregation was considered a random process by which misfolded proteins stick to each other to form inclusions<ref name=treusch>{{cite journal |doi=10.4161/cc.8.11.8503 |title=Amyloid deposits: Protection against toxic protein species? |date=2009 |last1=Treusch |first1=Sebastian |last2=Cyr |first2=Douglas M. |last3=Lindquist |first3=Susan |journal=Cell Cycle |volume=8 |issue=11 |pages=1668–74 |pmid=19411847|pmc=4451085 |url=http://dspace.mit.edu/bitstream/1721.1/54770/1/Lindquist1.pdf }}</ref> (imagine a bundle of hairs haphazardly piling up in a corner of a room). Moreover, protein aggregates were thought to be toxic agents and the cause for neuronal dysfunction and death. However, recent studies, using advanced methods (i.e. [[Fluorescence microscope|fluorescence microscopy]]), show that protein aggregation may actually be a tightly regulated, organized process, by which the cell protects itself from toxic proteins by sequestration to inclusion bodies.<ref name=Tyedmers>{{cite journal |doi=10.1038/nrm2993 |title=Cellular strategies for controlling protein aggregation |date=2010 |last1=Tyedmers |first1=Jens |last2=Mogk |first2=Axel |last3=Bukau |first3=Bernd |journal=Nature Reviews Molecular Cell Biology |volume=11 |issue=11 |pages=777–88 |pmid=20944667|s2cid=22449895 }}</ref> In 2008, Daniel Kaganovich working in the Frydman lab showed that [[eukaryotic]] cells sort [[misfolded proteins]] into two distinct inclusion bodies in a well-managed cellular process:<ref name=kaganovich>{{cite journal |doi=10.1038/nature07195 |title=Misfolded proteins partition between two distinct quality control compartments |date=2008 |last1=Kaganovich |first1=Daniel |last2=Kopito |first2=Ron |last3=Frydman |first3=Judith |journal=Nature |volume=454 |issue=7208 |pages=1088–95 |pmid=18756251 |pmc=2746971|bibcode = 2008Natur.454.1088K }}</ref> # The '''JUNQ''' (JUxta Nuclear Quality control compartment) # The '''IPOD''' (Insoluble Protein Deposit)
JUNQ and IPOD are [[evolutionarily conserved]], and are found in specific and defined cellular sites. Delivery of misfolded, aggregated proteins to JUNQ and IPOD require an intact [[cytoskeleton]] and specific cellular quality control components, such as [[Heat shock proteins|Heat Shock Proteins (HSPs)]].<ref name=specht>{{cite journal |doi=10.1083/jcb.201106037 |title=Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae |date=2011 |last1=Specht |first1=S. |last2=Miller |first2=S. B. M. |last3=Mogk |first3=A. |last4=Bukau |first4=B. |journal=The Journal of Cell Biology |volume=195 |issue=4 |pages=617–29 |pmid=22065637 |pmc=3257523}}</ref> The partition into the two distinct inclusion bodies is due to the different handling and processing of different kinds of [[misfolded proteins]] (e.g. [[ubiquitin]]ated vs. non-ubiquitinated proteins). Segregation of toxic protein aggregates into JUNQ and IPOD inclusion bodies is a means by which mammalian cells can be rejuvenated through asymmetric division.<ref name="pmid24843142">{{cite journal |vauthors=Ogrodnik M, Salmonowicz H, Brown R, Turkowska J, Sredniawa W, Pattabiraman S, Amen T, Abraham AC, Eichler N, Lyakhovetsky R, Kaganovich D | title=Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin | journal=[[Proceedings of the National Academy of Sciences of the United States of America]] | date=2014 | pmid=24843142 | doi=10.1073/pnas.1324035111 | pmc=4050583 | volume=111 | issue=22 | pages=8049–54| bibcode=2014PNAS..111.8049O| doi-access=free }}</ref>
Thus, the discovery of JUNQ and IPOD provided a new striking perspective of how cells manage misfolded aggregated proteins and gave convincing proof that [[protein aggregation]] is a non-random, well regulated and controlled cellular process. Furthermore, the discovery of JUNQ and IPOD suggested that in addition to temporal quality control (i.e. time dependent administration of damaged proteins) cells exploit [[homeostasis]] spatially:<ref name="spatial quality control">{{cite journal |doi=10.1098/rstb.2010.0282 |title=Spatial protein quality control and the evolution of lineage-specific ageing |date=2010 |last1=Nystrom |first1=T. |journal=Philosophical Transactions of the Royal Society B: Biological Sciences |volume=366 |issue=1561 |pmid=21115532 |pages=71–5 |pmc=3001311}}</ref> If degradation is unavailable, protection of the cellular environment from a [[misfolded protein]] is accomplished by its sequestration to an aggregate inclusion.{{cn|date=December 2021}}
== Background == To function properly, most [[proteins]] must preserve a low-energy, three-dimensional structure known as the [[native state]]. The stability of a protein is tightly regulated through all its life stages: from cradle, as it is synthesized at the [[ribosome]], through [[Protein folding|folding or assembly]], till grave – when the protein is degraded and cleared from the cellular environment.<ref name=morimoto>{{cite journal |doi=10.1093/gerona/gln071 |title=Protein Homeostasis and Aging: Taking Care of Proteins from the Cradle to the Grave |date=2009 |last1=Morimoto |first1=R. I. |last2=Cuervo |first2=A. M. |journal=The Journals of Gerontology Series A: Biological Sciences and Medical Sciences |volume=64A |issue=2 |pmid=19228787 |pages=167–70 |pmc=2655025}}</ref> Protein [[homeostasis]] (proteostasis),<ref name=powers>{{cite journal |doi=10.1146/annurev.biochem.052308.114844 |title=Biological and Chemical Approaches to Diseases of Proteostasis Deficiency |date=2009 |last1=Powers |first1=Evan T. |last2=Morimoto |first2=Richard I. |last3=Dillin |first3=Andrew |last4=Kelly |first4=Jeffery W. |last5=Balch |first5=William E. |journal=Annual Review of Biochemistry |volume=78 |pages=959–91 |pmid=19298183}}</ref> results from the coordinated action of the different arms of the cellular quality control system: [[molecular chaperones]], [[proteases]] and other regulatory factors. Hence, cellular viability depends on timely and efficient management of misfolded proteins. Such management, by the quality control machinery, includes recognition of the misfolded protein by [[chaperone (protein)|chaperone]]s and [[E3 ligase]]s, [[ubiquitin]]ation and [[Proteolysis|degradation]].{{cn|date=December 2021}}
Proteostasis collapse, due to damage, stress, [[mutations]], and [[aging]], has been implicated as a basis for a large number of common human disorders, such as [[neurodegenerative diseases]].<ref name="ben zvi">{{cite journal |doi=10.1073/pnas.0902882106 |title=Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging |date=2009 |last1=Ben-Zvi |first1=A. |last2=Miller |first2=E. A. |last3=Morimoto |first3=R. I. |journal=Proceedings of the National Academy of Sciences |volume=106 |issue=35 |bibcode=2009PNAS..10614914B |jstor=40484529 |pmid=19706382 |pages=14914–9 |pmc=2736453|doi-access=free }}</ref> Although caused by different kinds of mutated proteins (e.g. in [[Huntington's disease]] – the protein [[Huntingtin]]) and disruptive to distinct tissues (e.g. in [[Huntington's disease]] – the [[striatum]]), such diseases share a common feature: accumulation of misfolded proteins in [[inclusion bodies]]. Thus, it was thought that the inclusion bodies are the cause of such diseases. However, the nature and characteristics of those intra-cellular inclusion bodies stayed elusive. Different kinds of proteins (e.g. [[prions]], [[ERAD]] [[Substrate (biology)|substrates]]) were reported to form different kinds of inclusion bodies (e.g. [[aggresomes]], [[amyloids]]), yet it remained obscure if those observations combine into one and relate to the same sub-cellular site. Moreover, the pathways leading to inclusion formation and the involvement of the cellular protein quality control machinery were undefined and unknown. Thus, a systematic study providing a comprehensive understanding of [[protein aggregation]] and [[inclusion bodies]] was required. The discovery of JUNQ and IPOD<ref name= kaganovich /> suggested new insights of how the cell manages different kinds of misfolded proteins and offered a novel framework for putting together the great puzzle of [[protein aggregation]].<ref name=Tyedmers /> [[File:A cell harboring JUNQ and IPOD inclusions.png|thumb|upright=1.3|Eukaryote cells sort misfolded proteins, based on their ubiquitination state, into two quality control compartments:
1. JUNQ (green), which is tethered to the [[Cell nucleus|nucleus]] (orange) 2. IPOD(green), which is tethered to the [[vacuole]] (black shadow)]]
== Discovery == The fate of [[misfolded proteins]] and the process leading to the formation of aggregate inclusions, were initially studied using [[biochemical]] methods (e.g. [[western blotting]]).{{cn|date=December 2021}}
Deeper insights into the biological process of protein quality control and aggregation was made possible by a novel approach to looking at this problem, termed "Live Cell Imaging".<ref>{{Cite web | url=http://www.microscopyu.com/articles/livecellimaging/ |title = Live-Cell Imaging}}</ref>
Live cell imaging enables [[in vivo]] tracking of proteins in space and time, in their natural endogenous environment. Thus, such a method provides more information about the dynamics and stages of biological events and processes. The method takes advantage of the easily detectable [[fluorescent protein]]s fused to a protein of interest, which can then be followed inside a cell using a [[fluorescence microscope]]. The cell may then be treated by a perturbation of interest (e.g. a drug, expression of a [[misfolded protein]]), and various properties of the fluorescently tagged protein can be assayed using [[time-lapse microscopy]]: # Changes of the fluorescence level indicates changes of expression levels (i.e. higher levels = upregulation of a protein) # Changes of localization (e.g. entrance of a protein from the [[cytosol]] to the [[Cell nucleus|nucleus]]) # Solubility (e.g. by using the [[Fluorescence recovery after photobleaching|FRAP]] assay<ref name="flip and fraps">{{cite journal |doi=10.1126/science.1082520 |title=Development and Use of Fluorescent Protein Markers in Living Cells |date=2003 |last1=Lippincott-Schwartz |first1=J. |journal=Science |volume=300 |issue=5616 |pages=87–91 |pmid=12677058 |last2=Patterson |first2=GH|bibcode = 2003Sci...300...87L |s2cid=7831723 |url=https://zenodo.org/record/1230824 }}</ref>) # Interplay with the intracellular environment (e.g. by using the [[Fluorescence loss in photobleaching|FLIP]] assay<ref name="flip and fraps" />)
In order to monitor the fate of [[cytosolic]] [[misfolded proteins]] ''[[in vivo]]'', a [[plasmid]] carrying a [[Green fluorescent protein|GFP]] tagged folding reporter was [[Molecular cloning|cloned]]. The folding reporter, a model protein for aggregation, was a Ubc9 [[SUMO enzymes|(SUMO-conjugating enzyme)]] [[mutant]] (UBC9ts), harboring a [[missense mutation]] ([[Proteinogenic amino acid|Y68L]]) with a temperature- sensitive (ts) phenotype.<ref name=ubc9>{{cite journal |doi=10.1074/jbc.271.42.25790 |title=A Yeast Ubc9 Mutant Protein with Temperature-sensitive in Vivo Function is Subject to Conditional Proteolysis by a Ubiquitin- and Proteasome-dependent Pathway |date=1996 |last1=Seufert |first1=W. |journal=Journal of Biological Chemistry |volume=271 |issue=42 |pages=25790–6 |pmid=8824207 |last2=Seufert |first2=W|doi-access=free }}</ref><ref name="ubc9 2">{{cite journal |doi=10.1006/abio.1999.4190 |title=Characterization of a Temperature-Sensitive Mutant of a Ubiquitin-Conjugating Enzyme and Its Use as a Heat-Inducible Degradation Signal |date=1999 |last1=Tongaonkar |first1=Prasad |last2=Beck |first2=Konrad |last3=Shinde |first3=Ujwal P. |last4=Madura |first4=Kiran |journal=Analytical Biochemistry |volume=272 |issue=2 |pages=263–9 |pmid=10415098}}</ref> The marginally stable Ubc9ts is fully functional under [[physiological]] permissive conditions (25 °C) due to active cellular [[chaperone (protein)|chaperone]]s. The GFP–Ubc9ts was [[Transformation (genetics)|transformed]] into [[yeast]] and visualized using a [[fluorescence microscope]].{{cn|date=December 2021}}
Monitoring the folding sensor GFP–Ubc9ts was thought to indicate the cellular proteostasis, and to assay the ability of the cellular protein quality control system to deal with various kinds of stress. It was then observed that under normal conditions, GFP–Ubc9ts is diffused in the [[Cell nucleus|nucleus]] and in the [[cytosol]]. However, upon [[heat shock]], GFP–Ubc9ts formed cytosolic punctate structures. Strikingly, when the [[proteasome]] was impaired and clearance of the misfolded protein by [[Proteolysis|degradation]] was blocked, two distinct [[cytosolic]] inclusions were observed to be formed. Standard and conservative biochemical methods, such as [[cell fractionation]] and [[western blotting]] would not have revealed the partition into the two types of cytosolic aggregates.{{cn|date=December 2021}}
The two detected inclusions were shown to be [[evolutionarily conserved]] quality control compartments, with different characteristics and distinct functions. They were named JUNQ (JUxta Nuclear Quality control compartment) and IPOD (Insoluble Protein Deposit),<ref name= kaganovich /> and represent two cellular pathways for the sequestration and management of aggregation prone, potentially toxic proteins.{{cn|date=December 2021}}
Partition of quality control substrates (i.e. [[misfolded proteins]]) to either compartment depends on their [[ubiquitination]] status and aggregation state (i.e. solubility):{{cn|date=December 2021}}
Proteins that are ubiquitinated are delivered to the JUNQ, where they are processed for degradation by the [[proteasome]]. Misfolded proteins that are not ubiquitinated and terminally aggregated are sequestered to the IPOD.
Thus, the sub-cellular location of a [[misfolded protein]] (i.e. in the JUNQ or in the IPOD) provides information about its interaction with the cellular protein quality control machinery (e.g. its [[E3 ligase]]).
==JUNQ== '''JUNQ''' is the '''JU'''xta '''N'''uclear '''Q'''uality control compartment.
[[File:JUNQ (green) tethered to the nucleus (orange).tif|thumb|upright=1.3|A JUNQ inclusion viewed by a ubiquitinated VHL protein(green), is tethered to the [[Cell nucleus|nucleus]] (orange).]]
===Significance=== To maintain cellular homeostasis, the cellular quality control system must distinguish between folded and misfolded proteins. A misfolded protein will be recognized and tightly taken care of by either refolding or ubiquitination and proteasomal degradation.
However, cellular increase of misfolded protein loads, due to various kinds of stresses (e.g. [[heat shock]]), may saturate and exhaust the quality control machinery. In such cases, degradation of [[misfolded proteins]] is unavailable, and a second line of active cellular defense mechanism must be executed: directing [[misfolded proteins]] to specific cellular sites.<ref name=Tyedmers />
The JUNQ serves as such a sequestration site. It was shown<ref name= kaganovich /> that when the proteasome is impaired (e.g. by low expression levels of the [[proteasome]] subunit RPN11), [[ubiquitinated]] [[misfolded proteins]] are sorted into the JUNQ. Upon recovery from stress conditions (e.g. recovery from [[heat shock]] at a permissive temperature), [[misfolded proteins]] that accumulate in the JUNQ may be either refolded by the cellular [[Molecular chaperone|chaperone]] machinery, or degraded by the [[26S proteasome]]. Thus, the sequestration of a protein to the JUNQ is reversible.{{cn|date=December 2021}}
===Properties=== The JUNQ is a non- [[membrane]] bound cellular site located in a margin of the nucleus, near the [[endoplasmic reticulum]]. [[Fluorescence recovery after photobleaching|FRAP]] and [[Fluorescence loss in photobleaching|FLIP]] assays revealed that proteins in the JUNQ are soluble and exchange with the [[cytosol]], suggesting that the JUNQ has a dynamic structure.
Delivery to the JUNQ depends on [[molecular chaperones]] and [[co-chaperone]]s and on the [[actin]] [[cytoskeleton]].<ref name=specht /> [[Misfolded proteins]] must be [[ubiquitinated]] to be sorted to the JUNQ. If [[ubiquitination]] is blocked, a [[misfolded protein]] will be directed to the IPOD inclusion. [[Misfolded protein]] accumulation recruits 26S proteasomes to the JUNQ.
==IPOD== '''IPOD''' is the '''I'''nsoluble '''P'''rotein '''D'''eposit compartment.
[[File:IPOD (red) tethered to the vacuole (green).tif|thumb|upright=1.3|An IPOD inclusion viewed by a non-ubiquitinated VHL protein(red), tethered to the [[vacuole]] (green).]]
===Significance=== It is becoming more evident that the cellular capacity to maintain [[proteostasis]]<ref name=powers /> declines with age,<ref name="ben zvi" /> thereby causing the late onset of [[neurodegenerative]] diseases. In such diseases (e.g. [[Huntington's disease]]), a [[mutated]] protein misfolds and becomes toxic to the cellular environment by various ways such as denaturating cytosolic proteins.<ref name="urea like">{{cite journal |doi=10.1016/j.febslet.2010.12.023 |title=Polyglutamine shows a urea-like affinity for unfolded cytosolic protein |date=2011 |last1=England |first1=Jeremy L. |last2=Kaganovich |first2=Daniel |journal=FEBS Letters |volume=585 |issue=2 |pages=381–4 |pmid=21176779|s2cid=348468 |doi-access=free |bibcode=2011FEBSL.585..381E }}</ref> Incompetent of degrading those toxic species, the cell must isolate them to avoid their hazardous interaction with the cellular [[proteome]]. The IPOD was shown<ref name=kaganovich /> to be the sub-cellular site to which toxic [[amyloidogenic]] proteins are sequestered to, hereby serving as a protective quality control compartment.
In addition, it was suggested by the [[Susan Lindquist|Lindquist group]], that the IPOD is the site where [[yeast prions]] undergo a maturation process.<ref name="ancient compartment">{{cite journal |doi=10.1073/pnas.1003895107 |title=Prion induction involves an ancient system for the sequestration of aggregated proteins and heritable changes in prion fragmentation |date=2010 |last1=Tyedmers |first1=J. |last2=Treusch |first2=S. |last3=Dong |first3=J. |last4=McCaffery |first4=J. M. |last5=Bevis |first5=B. |last6=Lindquist |first6=S. |journal=Proceedings of the National Academy of Sciences |volume=107 |issue=19 |bibcode=2010PNAS..107.8633T |pmid=20421488 |pages=8633–8 |pmc=2889312|doi-access=free }}</ref> Thus, the IPOD may serve not only as a sequestration site, but also as a functional compartment.<ref name="ancient compartment" />
===Properties=== The IPOD is a non- [[membrane]] bound cellular site, which in yeast is located by the [[vacuole]]. [[Fluorescence recovery after photobleaching|FRAP]] and [[Fluorescence loss in photobleaching|FLIP]] assays revealed that proteins in the IPOD are tightly packed, in-soluble and do not exchange with the [[cytosol]]. [[Amyloidogenic]] proteins, such as the [[Huntingtin]] protein, are the IPOD's substrates.{{cn|date=December 2021}}
[[Misfolded proteins]] must be non-[[ubiquitinated]] to be sorted to the IPOD. Ubiquitination of an otherwise IPOD substrate, such as the RNQ1 [[fungal prion]], will result in its sequestration in the JUNQ inclusion.{{cn|date=December 2021}}
Upon accumulation of [[misfolded proteins]], the disaggregase [[Molecular chaperone|chaperone]], [[AAA protein]] HSP104, localizes to the IPOD. It is yet to be determined if HSP104 functions in the IPOD or is simply sequestered there being hooked to a substrate.
The pre-autophagosomal structure (PAS) is localized by the IPOD.<ref name=pas>{{cite journal |doi=10.1093/emboj/20.21.5971 |title=The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation |date=2001 |last1=Suzuki |first1=K. |journal=The EMBO Journal |volume=20 |issue=21 |pages=5971–81 |pmid=11689437 |last2=Kirisako |first2=T |last3=Kamada |first3=Y |last4=Mizushima |first4=N |last5=Noda |first5=T |last6=Ohsumi |first6=Y |pmc=125692}}</ref> However, it was not shown that IPOD substrates are delivered to the vacuole, and so the link between the IPOD and [[autophagy]] is yet to be determined.<ref name=kaganovich />
==See also== * [[Confocal laser scanning microscopy]] * [[Protein aggregation]]
==References== {{Reflist}}
== External links == * [http://www.kaganovichlab.com/research.html Protein aggregation] * [http://www.englandlab.com/protein-folding.html Protein folding] * [http://www.microscopyu.com/articles/livecellimaging/ Live cell imaging] * [https://www.youtube.com/watch?v=FPCqvdsnJwQ Jennifer Lippincott-Schwartz : Intracellular Fluorescent Imaging: An Introduction] * [https://www.youtube.com/watch?v=_Q0oOcZminY Susan Lindquist (MIT) : Protein Folding and Prions]
[[Category:Alzheimer's disease]] [[Category:Neurodegenerative disorders]] [[Category:Neurological disorders]] [[Category:Structural proteins]] [[Category:Huntington's disease| ]] [[Category:Proteins]] [[Category:Protein complexes]] [[Category:Protein structure]] [[Category:Organelles]] [[Category:Microscopy]] [[Category:Fluorescence]] [[Category:Cell imaging]] [[Category:Yeasts]]