Vladimir N. Gene Regulatory Networks and their Role in Protein Homeostasis 4 Lectures Network motifs: basic building blocks of biological networks Network motifs: basic building blocks of biological networks. Structure, evolution and dynamics of gene regulatory netw Structure, evolution and dynamics of gene regulatory networks.
Computational methods in analysis of gene regulation and Computational methods in analysis of gene regulation and protein interactions. Protein-protein interaction networks Protein-protein interaction networks. Peter Csermely Semmelweis University, Hungary. Protein Homeostasis in the Endoplasmic Reticulum 6 Lectures Role of calnexin and calreticulin in protein homeostasis Role of calnexin and calreticulin in protein homeostasis within the endoplasmic reticulum.
David B. Williams University of Toronto, Canada. The unfolded protein response The unfolded protein response. Kazutoshi Mori Kyoto University, Japan. Role of ER stress in cystic fibrosis airway inflammation Role of ER stress in cystic fibrosis airway inflammation. The recognition of misfolded glycoproteins in the endopla The recognition of misfolded glycoproteins in the endoplasmic reticulum.
David Y. Thomas McGill University, Canada. Chaperone systems of the endoplasmic reticulum Chaperone systems of the endoplasmic reticulum. Linda M. Next, the substrate can be transferred to another chaperone system, such as the chaperonins, where folding takes place and a three-dimensional structure is acquired reviewed in [ 12 , 16 , 17 ].
When misfolded proteins accumulate, unfolded protein responses can increase the levels of chaperones, which are then able to restore the proteins to their properly folded form Fig. Such an accumulation of misfolded protein is just one of the types of stress that can trigger unfolded protein responses. Unfolded protein responses are mechanisms that are highly conserved from yeast to humans and that are induced upon environmental and physiological stress, such as thermal or oxidative stress reviewed in [ 22 — 24 ].
In one of these pathways thought to respond to the accumulation misfolded proteins in the cytosol, heat shock factor 1 HSF-1 acts as a master transcriptional regulator. HSF-1 is activated upon phosphorylation, after which it translocates from the cytosol to the nucleus to bind to the so-called heat shock elements, thereby upregulating the transcription of heat shock genes.
These genes are then translated into proteins that assist in the refolding of misfolded proteins into functionally active proteins, in preventing unspecific interactions, or in mediating their degradation Fig. Another strategy used by the cell to restore protein homeostasis is the unfolded protein response that is associated with the endoplasmic reticulum ER Fig. The ER is the organelle where proteins enter the secretory pathway to acquire post-translational modifications, after which they are delivered to their corresponding organelle, fixed in the plasma membrane or shuttled outside of the cell to perform their function [ 27 ].
Firstly, IRE1 is a transmembrane protein kinase that activates itself by auto-phosphorylation and mediates splicing of Hac1 in yeast and XBP-1 in eukaryotes [ 28 — 32 ]. IRE1 is known to promote the transcription of three groups of genes: stress-responsive genes including molecular chaperones and folding enzymes, genes involved in ERAD and genes involved in ER trafficking [ 33 — 35 ]. Secondly, ATF-6 is a transmembrane protein with a transcription factor domain leucine zipper that translocates from the ER lumen to the Golgi apparatus to be cleaved by proteases [ 36 , 37 ].
This proteolysis releases the ATF-6 cytosolic fragment, which then enters the nucleus to induce the transcription of ER-resident chaperones and the transcription factor XBP-1, thereby increasing ER protein quality control capacity [ 29 , 37 — 39 ]. Thirdly, PERK is a transmembrane kinase protein that phosphorylates the alpha-subunit of the eukaryotic translation initiation factor 2a eIF2a , thus preventing the binding of the initiator tRNA Met to the ribosomal complex, necessary for translation initiation [ 40 — 42 ].
This results in an overall reduction in protein synthesis, thereby attenuating the accumulation of misfolded proteins at the ER. If an aberrant protein cannot be folded back into its native state by the molecular chaperones, then it can be eliminated by two proteolytic systems, the proteasome and autophagy Fig. In the degradation via the ERAD pathway, the ER cooperates tightly with the ubiquitin—proteasome system UPS to recognize, mark and traffic the misfolded proteins to the cytosol for degradation Fig.
The exact mechanisms that allow the cell to discriminate misfolded proteins from correctly folded proteins are not fully understood reviewed in [ 44 , 46 , 47 ]. An example that illustrates this recognition is the immunoglobulin binding protein BiP , an HSP70 chaperone that recognizes and binds to the hydrophobic regions of misfolded proteins, thereby preventing their aggregation [ 49 — 53 ].
These factors stimulate the ATPase activity of BiP and stabilize its binding to the misfolded protein [ 54 — 58 ]. The ERdj co-chaperones have also been shown to bind directly to unfolded proteins, thus maintaining them in a soluble state to be later recruited by BiP [ 48 , 59 ]. After the misfolded protein has been identified, it is poly-ubiquitinated to be subsequently targeted for degradation [ 60 — 62 ].
Ubiquitination is a sequential three-step process that marks proteins destined for the proteasome Fig. It starts with the activation of ubiquitin a small 76 amino acid protein by the activating enzyme E1, followed by binding of ubiquitin to the active site of the ubiquitin-carrier protein E2 and, finally, transfer of the ubiquitin molecule to the substrate in a reaction catalyzed by the ubiquitin protein ligase E3.
At least four ubiquitin molecules must be bound to the ERAD substrate for it to be later recognized by the proteasomal machinery [ 63 , 64 ]. Following this step, the misfolded proteins are delivered to the proteasome a process called retrotranslocation and the ubiquitin molecules are removed from the substrate prior to degradation by the deubiquitinating enzymes and recycled [ 65 — 67 ].
The proteasome is a barrel-shaped, multicatalytic proteinase where proteolysis occurs and proteins are cleaved into peptides 2—30 amino acid long [ 68 ]. It is the part of the cell that ensures protein and organelle turnover, where old cellular components are degraded and recycled molecules become available for cell metabolism [ 70 , 71 , 73 ]. For the purpose of this review, we discuss only the role of autophagy as a protein quality control system.
Autophagy can be classified into three categories: macroautophagy, microautophagy and chaperone-mediated autophagy CMA. In macroautophagy, a newly formed double membrane vesicle engulfs the cytosolic material, forming the autophagosome.
The autophagosome then fuses with an endosome or lysosome, giving rise to the autolysosome where degradation takes place through the action of hydrolytic enzymes Fig. The double membrane that surrounds the autophagosome is derived from the ER, the mitochondria or the plasma membrane [ 74 — 78 ]. In yeast, autophagy is a multi-step process that requires at least 37 autophagy-related ATG genes [ 79 — 89 ]. The majority of the ATG genes have shown to be functionally conserved in mammals [ 90 , 91 ].
In microautophagy, small molecules from the cytoplasm are internalized by the lysosome through invagination of its own membrane [ 70 , 73 ]. In contrast to autophagy and CMA, much less is known about microautophagy [ 92 ].
CMA differs from the former two forms of autophagy in that it does not involve membrane reorganization. Here, the substrate is unfolded before it is translocated into the lumen of the lysosome for degradation, which is assisted by Hsc73, an intralysosomal HSP70 chaperone [ 97 , 98 ]. Crosstalk exists between the UPS and autophagy. Chronic low-level proteasomal inhibition is known to be sufficient to activate autophagy, and it has been suggested that ubiquitinated proteins may also be eliminated through this pathway [ 99 — ].
It has also been proposed that macroautophagy may occur as a compensatory mechanism when either the UPS or CMA is impaired [ , ]. An alternative pathway for misfolded proteins is the sequestration into specialized protein quality control compartments where they can be either recovered or permanently sequestered Fig.
Distinct quality control compartments harbor different species of misfolded proteins and are evolutionary conserved from yeast to mammals [ , — , , ]. Ubiquitinated misfolded cytosolic proteins are assigned to the juxtanuclear quality control compartment JUNQ, Fig. These soluble, mobile misfolded proteins can subsequently be recovered by the molecular chaperone Hsp and either refolded back into functionally active proteins or degraded by the proteasomes localized nearby Fig. Non-ubiquitinated misfolded proteins—comprising amyloidogenic proteins—are redistributed to the insoluble protein deposit IPOD, Fig.
This compartment is localized at the cell periphery and is known to contain insoluble and immobile species, which are not recoverable and seem to remain terminally sequestered there Fig. Much research has focused on finding out whether the redistribution of misfolded proteins to these spatial cytosolic compartments is a random event or whether it depends on the concerted action of sorting factors.
Evidence suggests that the latter is the case, and that sorting factors interact with chaperones to deliver misfolded proteins to each compartment [ ]. For example, upon physiological stress, Btn2 a Hook family protein involved in linking organelles to microtubules was shown to associate either with the yeast small heat shock protein Hsp42 to assign misfolded proteins to the IPOD or with the chaperone Sis1 to guide misfolded proteins to the JUNQ [ , ].
Another type of cytosolic compartment—the aggresome—is localized at the microtubule organizing center MTOC and is formed when the proteasome is unable to clear misfolded proteins properly Fig.
Aggresome formation is accompanied by redistribution of vimentin, an intermediary filament that acquires a cage-like structure in the aggresome. Interestingly, the JUNQ shares several properties with the aggresome, including its perinuclear localization, and the presence of chaperones and ubiquitinated misfolded proteins [ , ].
It has also recently been shown to functionally associate with the MTOC and vimentin [ ]. Indeed, continuous accumulation of misfolded proteins in the JUNQ is thought to turn it in an aggresome over time [ ]. Similar structures to aggresomes are the so-called aggresome-like induced structures ALIS , which were originally discovered in dendritic cells but were later also found in other type of cells [ , ].
The ALIS is a transient structure with peripheral and juxtanuclear localization. It is induced under a wide variety of stress conditions e. ALIS substrates can also be cleared by the proteasome and lysosome [ ]. Cell division could be considered as yet another protein quality control system that sequesters misfolded, aggregated proteins reviewed in [ , ].
Studies in bacteria and yeast have shown that accumulation of protein aggregates reduces the fitness of these cells, a problem partially resolved by asymmetric division: these protein deposits are retained in the aging mother cell while the daughter cells are freed from damaged proteins, a process also known as replicative rejuvenation [ — ].
In budding yeast, it has been shown that misfolded proteins sorted either to the JUNQ or IPOD remain in the mother cell after asymmetric cell division, thus avoiding passage of these species onto the daughter cells [ ]. Follow-up work from the same group extended this observation to mammalian cells, where the JUNQ but not the IPOD continues to be inherited asymmetrically, thereby always freeing one of the two daughter cells from proteotoxicity [ ].
While much is now known about the sophisticated quality control mechanisms that the cell has evolved to ensure proper protein homeostasis, several questions remain to be answered. We know that the cell relies on the concerted action of chaperones to prevent an unfolded or misfolded protein interacting aberrantly with other proteins until it can be refolded back into its native state.
In case this is not possible, the aberrant protein is sent to be degraded via the ubiquitin—proteasome system or by autophagy. However, it is still not known how the cell chooses one mechanism of degradation over the other or whether the two mechanisms occur simultaneously. Another unknown relates to protein compartmentalization—yet another strategy for putting away proteins that need to be degraded or permanently sequestered.
It has not yet been established how the cell can differentiate between degradable and non-degradable proteins and shuttle them to different subcellular compartments. Finally, another important question is how protein quality control changes during aging. Aging itself may be the contributing factor for progressive deterioration of protein homeostasis, impairing the ability of the protein quality control system to handle the equilibrium between protein folding and degradation. The effects of progressive deterioration of protein homeostasis are thought to play a role in age-related neurodegenerative diseases.
In these diseases, it is not yet clear why proteins accumulate into aggregates and how this relates to pathogenesis. Protein aggregation and its relationship to aging and neurodegeneration have also been widely studied in animal models. This could lead to the progressive accumulation of cytotoxic aggregation-prone disease proteins that cannot be cleared, ultimately resulting in toxicity and cell death [ , — ].
In the roundworm Caenorhabditis elegans, a model organism much used to study aging, protein aggregation has been shown to occur during aging and to affect the lifespan of the organism [ — ]. As previously discussed, when a protein misfolds it exposes its aggregation-prone domains to the cellular environment—domains that would otherwise be structurally concealed—thereby facilitating the likelihood of aberrant interactions with other proteins, potentially leading to proteotoxicity.
The type of aggregates that are formed varies for different neurodegenerative diseases. Frontotemporal lobar degeneration with fused in sarcoma is an example of a neurodegenerative disease that is characterized by the presence of amorphous, non-amyloidogenic aggregates [ , ], also reviewed in [ ].
On the other hand, the common neuropathological feature of PD, AD and HD is the presence of an aggregation-prone disease protein that acquires amyloidogenic properties, causing it to form intracellular amyloid aggregates or extracellular amyloid plaques in the brains of patients reviewed in [ , , ].
The amyloids present in these neurodegenerative diseases can be distinguished from other amorphous, unstructured aggregates because they are organized, insoluble fibrils with a cross-beta structure and because they can be detected by specific amyloid-binding dyes, namely Congo red and thioflavin T reviewed in [ , ]. It is interesting to note that—despite their differences in amino acid sequence and function—several unrelated aggregation-prone disease proteins have one thing in common: in disease they are present as amyloid.
This suggests that their ability to form amyloid is related to disease and that they may cause proteotoxicity in a similar manner. In vitro studies have made clear that virtually any protein can form amyloid fibrils under certain conditions. Such conditions include low pH, high temperature and high pressure [ — ].
Native proteins are known to exist in equilibrium with their partially unfolded state, and when they are destabilized by certain conditions or mutations, the equilibrium shifts towards amyloid formation. Predicting aggregation-prone regions in proteins is now possible using bioinformatic tools. Examples of such tools are TANGO, which can specifically identify regions prone to form beta sheets, and Waltz, which can distinguish between amyloid sequences and amorphous beta-sheet aggregates [ , ].
A proposed mechanism for amyloid formation is depicted in Fig. Most of our understanding of this pathway has come not only from in vitro studies of aggregation-prone proteins, including amyloid-beta seen in AD and alpha-synuclein seen in PD but also from studies of globular proteins, including human lysozyme, superoxide dismutase 1, transthyretin and the acylphosphatase from the archaea Sulfolobus solfataricus reviewed in [ 17 , , , , ].
One common step of amyloid formation appears to be the conversion of the monomeric, native state protein into an oligomeric intermediate state Fig. An oligomer is a small and transient cluster of protein molecules that has no fibrillar structure and is of low molecular weight [ — ]. These oligomers can then form protofibrils, which are fibrils 6 to 8 nm in diameter, about nm in length and known to contain beta sheets detectable by Congo red and thioflavin T staining Fig.
Protofibrils can then convert into amyloid fibrils Fig. Of all these aggregation intermediates, it is currently thought that the early ones are cytotoxic and that aggregation may be a neuroprotective response to permanently sequester these intermediates, thereby preventing potentially toxic interactions with other proteins in the cellular milieu [ — ]. In support of this hypothesis, it has been shown that proteins rich in beta-sheet structures aggregate with newly synthesized proteins that have not yet become folded or with intrinsically unfolded proteins, thereby reducing the availability of these proteins to perform their normal function [ ].
Further evidence demonstrating that oligomeric or protofibrillar forms of aggregation-prone disease proteins contribute to cell toxicity and death is reviewed elsewhere [ , , — ]. Proposed mechanism for amyloid formation.
A protein loses its monomeric native state by conversion into an oligomer which can grow further into amyloidogenic fibrils and ultimately into insoluble amyloid aggregates. In a nutshell, the amyloid pathway has only just started to be described and it is not fully understood how protein aggregation correlates with disease. At the clinicopathological level, it is striking that there are individuals with high AD pathology i.
This fact makes it difficult to discern what are the boundaries between normal aging and disease. At the cellular and molecular level, what structural properties do aggregation-prone proteins acquire that make them toxic? This question is further complicated by the fact that aggregation-prone proteins such as amyloid-beta, huntingtin or alpha-synuclein do not share sequence, structure or function.
A second question is that of how long neuronal cells can deal with these aggregation-prone proteins. And is their slow accumulation in the brain a reflection of an impaired protein quality control system? Finally, the majority of our knowledge about aggregation intermediates has come from in vitro studies. It remains to be shown whether oligomeric and fibrillar species exist in vivo and what their relevance to pathogenesis is.
The current understanding of how protein misfolding and aggregation contributes to neurodegeneration is far from complete. Whether different immune cell types or inflammatory conditions, including tumor immunity, are regulated by the uptake or exclusion of specific amino acids, or the preferential engagement of amino acid catabolic or anabolic pathways, has not been studied in deep. Moreover, the role of substrate competition for each amino acid transporter has not been assessed in immune responses.
Considering that organisms employ multiple homeostatic mechanisms in vivo to maintain extracellular amino acid levels, it is critical to understand how amino acid metabolism impacts immunity in vivo.
For this reason, further studies focusing on amino acid metabolism in acute and chronic inflammatory conditions are required. In this Research Topic, we welcome authors to submit Original Research, Mini Reviews and Perspective articles focusing on: 1 Regulation of amino acid transporters and amino acid-mediated metabolic pathways to control adaptive and innate immune responses, including immunity against tumors. Important Note : All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements.
Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review. In contrast, Klinked polyubiquitination modifies diverse protein functions including DNA repair and endocytosis but does not lead to proteasomal degradation [11,12].
However, it is still not fully understood how different types of polyubiquitin chains are decoded in a cellular system yet. Deubiquitinating enzymes DUBs play a role in removing ubiquitin molecules from substrates and have diverse roles in cellular processes [13]. It has been suggested that approximately DUBs are encoded in the human genome [2], and they are classified into distinct subfamilies depending upon the organization of the catalytic domain.
In general, they have been grouped into two main classes: cysteine protease and metalloprotease [14]. Dysregulation of DUBs is becoming evident that they can be causative in a number of human diseases including cancer, and immune and neurodegenerative diseases [18].
Therefore, DUBs have been good targets to develop therapeutic drugs especially for diseases like cancer and immune diseases.
And Dub-2 knock-out mice showed embryonic lethality and growth inhibition [25]. In addition, DUB-3 identified in human chorionic villi is induced by IL-4 and IL-6 and is involved in the regulation of cell growth and survival []. However, most of their substrates for these cytokine-inducible DUBs have not been found yet and their deubiquitinating enzyme activity has been mostly tested only in vitro.
Since the enzymatic activity of these DUBs has been tested in vitro with the long period of induction to cleave the ubiquitin, it is not possible to know how fast the enzymatic activisty appears. Spatial and temporal elimination of misfolded and damaged proteins is required to prevent the accumulation of toxic proteins, which can lead to diverse diseases including cancer and immune diseases [7]. However, it is not clear how a cell determines whether proteins targets should be degraded by the UPS or not.
Given the significance of ubiquitin signaling in human diseases, important directions for future studies will include the analysis of underlying mechanisms that regulate the UPS for developing therapeutic implications for cancer and other immune diseases. Even though cytokine-inducible DUBs are known to be involved in the regulation of cell proliferation and apoptosis, their DUB inhibitors have not been identified or developed for therapeutic purposes yet.
Therefore, important directions for future studies will include identification of their substrates and underlying molecular mechanisms, and delineation of their molecular structures to provide us a novel way of developing therapeutic drugs especially for disease like cancer and immune diseases.
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