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Abnormal polypeptides that escape proteasome-dependent degradation and aggregate in cytosol can be transported via microtubules to an aggresome, a recently discovered organelle where aggregated proteins are stored or degraded by autophagy. We used synphilin 1, a protein implicated in Parkinson disease, as a model to study mechanisms of aggresome formation. When expressed in naïve HEK293 cells, synphilin 1 forms multiple small highly mobile aggregates. However, proteasome or Hsp90 inhibitiondoi:10.1074/jbc.m802216200 pmid:18635553 fatcat:4vvbfeaq2bdovdughj34ppniqi
more »... dly triggered their translocation into the aggresome, and surprisingly, this response was independent on the expression level of synphilin 1. Therefore, aggresome formation, but not aggregation of synphilin 1, represents a special cellular response to a failure of the proteasome/chaperone machinery. Importantly, translocation to aggresomes required a special aggresome-targeting signal within the sequence of synphilin 1, an ankyrin-like repeat domain. On the other hand, formation of multiple small aggregates required an entirely different segment within synphilin 1, indicating that aggregation and aggresome formation determinants can be separated genetically. Furthermore, substitution of the ankyrin-like repeat in synphilin 1 with an aggresome-targeting signal from huntingtin was sufficient for aggresome formation upon inhibition of the proteasome. Analogously, attachment of the ankyrin-like repeat to a huntingtin fragment lacking its aggresome-targeting signal promoted its transport to aggresomes. These findings indicate the existence of transferable signals that target aggregation-prone polypeptides to aggresomes.
Sherman) and P41 RR10888 and S10 15942 (to Catherine E. Costello). ...doi:10.4161/pri.1.2.4440 pmid:19164926 pmcid:PMC2634453 fatcat:exsqr7mu4rdf7cskg7bqvhnkhq
Molecular chaperones and the ubiquitin-proteasome system (UPS) play an important role in handling soluble abnormal polypeptides that arise as a result of misfolding, damage or mutations. However, under certain conditions these systems fail to repair or destroy abnormal species, leading to formation of small cytoplasmic aggregates. Mechanisms of intracellular protein aggregation attract growing attention because of their relevance to adoi:10.1002/0471143030.cb0338s48 pmid:20853343 pmcid:PMC3422749 fatcat:pdhx6yqirnab5nhejbxtipgxh4
Sherman, manuscript in preparation. by guest on July 26, 2018 http://www.jbc.org/ Downloaded from J. Yaglom, unpublished data. 4 L. Monney, personal communication. ... .: 617-742-2010 (ext. 312); Fax: 617-523-6649; E-mail: firstname.lastname@example.org. 1 The abbreviations used are: JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; PARP, poly(ADP-ribose) polymerase ...doi:10.1074/jbc.273.11.6373 pmid:9497367 fatcat:72dpxaeh7jdcdkepyujxdf73ou
., 1997) which reduces the pool of newly synthesized misfolded, therefore aggregation prone, polypeptides (Conn and Qian, 2013; Sherman and Qian, 2013) . ...doi:10.7554/elife.39695 pmid:30716021 pmcid:PMC6361590 fatcat:jsq6h5epl5bfrpqwasjakeniu4
Failure in protein quality control can often lead to protein aggregation, yet in neuro-degenerative diseases, by the time aggregates can be seen, the cells have advanced well into the disease pathology. Here, we develop a quantitative imaging approach to study the protein aggregation process in living mammalian cells with unprecedented spatio-temporal resolution. We find that sub-diffractive precursor aggregates may form even in untreated cells, and their size distribution is exactly asdoi:10.1101/148395 fatcat:w6uq6u7txbh7vkqri67avau6zq
more »... d for a system undergoing a first order phase transition. Practically, this implies that as soon as aggregates reach a critical size (Rc = 162 ± 4 nm in untreated cells), they will spontaneously grow into large inclusions. Our data suggest that a previously uncharacterized, RuvBL1 dependent mechanism clears aggregates above the critical size. Our study unveils the existence of sub-diffractive aggregates in living cells; and the strong agreement between cellular data and a nucleation theory, based on first order phase transition, provides insight into regulatory steps in the early stages of aggregate formation in vivo.
.: 617-742-2010 (ext. 312); Fax: 617-523-6649; E-mail: email@example.com. 1 The abbreviations used are: JNK, Jun N-terminal kinase; MTT, 3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrasolium bromide ...doi:10.1074/jbc.272.29.18033 pmid:9218432 fatcat:d2zcsarq6nh7po6yecfp5lbvge
We demonstrate a novel method that enables one to measure the structure of highly reflecting fiber Bragg gratings. The method is based on measuring both the transmission and reflection spectra of the grating and applying an inverse-scattering algorithm. The use of the transmission spectrum significantly reduces the sensitivity of the reconstruction to measurement noise, and therefore it significantly decreases the measurement duration. We experimentally demonstrate our method for reconstructingdoi:10.1364/ol.32.000457 pmid:17392886 fatcat:zjjr32n2njau5pdqdfgx4srsdi
more »... the structure of an apodized grating with a reflectivity of 99.91%.
Cell protection from stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from stresses which do not cause direct protein damage, e.g. somedoi:10.1016/s0014-5793(98)01242-3 pmid:9821948 fatcat:6llde26jjvf5lcvpgjjkvrljvm
more »... c agents. These stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a stress-activated protein kinase, JNK, an early component of stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by stresses and subsequently to a higher rate of apoptotic death. z 1998 Federation of European Biochemical Societies.
Abnormal proteins, which escape chaperonemediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH 2 -terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulatesdoi:10.1083/jcb.153.4.851 pmid:11352944 pmcid:PMC2192371 fatcat:ox32rh7sgvc6fbdkuth5lvs624
more »... l stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.
Current strategies to alleviate protein misfolding include manipulation of chaperones, proteasomes, or autophagy. Results: Mild translation inhibition disproportionally blocked production of misfolded proteins and improved mutant CFTR function. Conclusion: Slowing down translation improves folding of newly synthesized proteins in mammalian cells and recovers mutant protein function. Significance: Attenuation of translation could be a novel approach to treatment of protein-misfolding disorders.doi:10.1074/jbc.m112.397307 pmid:22902621 pmcid:PMC3464534 fatcat:4j47rtxkuzg3dppupqnmbypaey
more »... rotein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.
INTRODUCTION. A major inducible heat shock protein Hsp70 (Hsp72) is known to protect cells from diverse apoptosis-inducing stimuli but the mechanism of the protection has not been established yet (1,2). We have previously found that Hsp70 may inhibit apoptotic signal transduction at an early step, via suppression of stress kinase JNK (3); in addition, recent data indicate that Hsp70 may block directly apoptosome formation and caspase-9/3 activation (4,5). However, besides "classical"doi:10.1100/tsw.2001.161 pmid:30147477 fatcat:ti6utje4v5dvffil6dfyb5mafq
more »... endent apoptosis, there is a programmed cell death that is caspase-independent. Here we studied whether Hsp70 can protect from such type of cell death and what is the mechanism of the protection. METHODS. To increase levels of Hsp70 we used adenovirus expressing human Hsp72 under control of tet-promoter, and adenovirus expressing dominant-negative mutant of SEK1 was used to inhibit JNK activation. Cell death was assessed by acridine orange/ethidium bromide staining under fluorescent microscope, or by poly-ADP ribose (PARP) cleavage and caspase-3 activation by immunoblotting. Activities of MAP kinases (ERK1/2, p38, and JNK) were measured by immunoblotting with antibodies to phosphorylated (activated) form of the kinases. RESULTS. Although the protective effect of Hsp70 against caspase-dependent apoptosis is well established, little is known about its role in caspase-independent cell death. We studied the effect of Hsp70 in two models of caspase-independent cell death: heat-induced killing of normal human fibroblasts and death of myogenic cells after transient ATP depletion. Morphology of heat-treated IMR90 diploid fibroblasts resembled that of apoptotic cells (e.g., after TNF or staurosporine treatment), and the plasma membrane remained intact. Morphology of H9c2 myogenic cells after transient ATP depletion was also similar to that of apoptotic cells, but some cells lost plasma membrane integrity. Both forms of cell death were not accompanied either by caspase-3 activation or PARP cleavage. Moreover, pancaspase inhibitor zVAD-fmk did not protect either from heat-induced killing of fibroblasts or ischemic death of H9c2 cells. Both heating and transient energy deprivation activated several MAP kinases, Erk1/2, p38, and JNK in these cells. As with classical apoptosis, inhibition of "survival" kinase, Erk1/2, decreased cell survival in both models. In contrast, inhibition of JNK (but not p38) almost completely protected from heat-induced killing of fibroblasts and markedly reduced death of H9c2 after energy deprivation. These types of caspase-independent cell death were effectively prevented by increased levels of Hsp70. Hsp70 overexpression greatly diminished activation of JNK after heat shock or transient energy deprivation; on the other hand, it did not increase activity of Erk1/2. Therefore, the protection of cells from the caspase-
Therefore, IB formation may be irrelevant to the neurodegeneration process or may even serve a protective role by capturing toxic soluble polyQ molecules (for review see Sherman and Goldberg, 2001) . ...doi:10.1083/jcb.200112104 pmid:12058016 pmcid:PMC2174031 fatcat:fcxwz3nhgzhwlmr4uoofodq4xa
Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out theSEK1gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylationdoi:10.1128/mcb.19.4.2547 pmid:10082520 pmcid:PMC84047 fatcat:6xlzrbzoyzcqpnkdrwo6jiy6pi
more »... n unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.
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