scholarly journals Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

2017 ◽  
Vol 114 (22) ◽  
pp. E4370-E4379 ◽  
Author(s):  
Jang-Hyun Oh ◽  
Ju-Yeon Hyun ◽  
Alexander Varshavsky
Keyword(s):  
Heat Shock Protein 90 ◽  
Mitotic Checkpoint ◽  
Protein Homeostasis ◽  
Wild Type ◽  
Binding Assays ◽  
C Terminus ◽  
Ubiquitin Binding ◽  
Hsp90 Chaperone ◽  
Split Ubiquitin

We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA Nα-terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway.

scholarly journals The Arg/N-degron pathway targets transcription factors and regulates specific genes

2020 ◽  
Vol 117 (49) ◽  
pp. 31094-31104
Author(s):  
Tri T. M. Vu ◽  
Dylan C. Mitchell ◽  
Steven P. Gygi ◽  
Alexander Varshavsky
Keyword(s):  
Ubiquitin Ligases ◽  
Actin Binding ◽  
Wild Type ◽  
Binding Assays ◽  
Double Knockout ◽  
Ubiquitin Binding ◽  
Specific Mrnas ◽  
Lactate Transporter ◽  
Split Ubiquitin ◽  
Mammalian Gene Expression

The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and β-adrenergic receptor ADRB2, are strongly (∼20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified ∼160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.

Saccharomyces cerevisiae Checkpoint Genes MEC1, RAD17 and RAD24 Are Required for Normal Meiotic Recombination Partner Choice

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 607-620 ◽  
Author(s):  
Jeremy M Grushcow ◽  
Teresa M Holzen ◽  
Ken J Park ◽  
Ted Weinert ◽  
Michael Lichten ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Mitotic Checkpoint ◽  
Partner Choice ◽  
Wild Type ◽  
Spore Viability ◽  
Checkpoint Signaling ◽  
Ectopic Recombination ◽  
Meiotic Progression ◽  
Checkpoint Genes

Abstract Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.

scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

scholarly journals The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10315-10328 ◽  
Author(s):  
Yukinori Minoshima ◽  
Tetsuya Hori ◽  
Masahiro Okada ◽  
Hiroshi Kimura ◽  
Tokuko Haraguchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Mitotic Checkpoint ◽  
Loss Of Function ◽  
Wild Type ◽  
Centromere Function ◽  
Dt40 Cells ◽  
Precise Role ◽  
Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

scholarly journals Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

2007 ◽  
Vol 179 (2) ◽  
pp. 255-267 ◽  
Author(s):  
Karthik Jeganathan ◽  
Liviu Malureanu ◽  
Darren J. Baker ◽  
Susan C. Abraham ◽  
Jan M. van Deursen
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Physiological Role ◽  
Mitotic Checkpoint ◽  
Critical Threshold ◽  
Wild Type ◽  
Checkpoint Protein ◽  
Chromosome Missegregation ◽  
Mitotic Checkpoint Protein

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

scholarly journals Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in Yersinia enterocolitica

2004 ◽  
Vol 186 (16) ◽  
pp. 5366-5375 ◽  
Author(s):  
Peter Burghout ◽  
Frank Beckers ◽  
Emmie de Wit ◽  
Ria van Boxtel ◽  
Guy R. Cornelis ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Yersinia Enterocolitica ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Protein Secretion System ◽  
Membrane Lipoprotein ◽  
Type Iii Protein Secretion

ABSTRACT The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

IGF Binding Protein 2 Supports the Cycling of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1604-1604
Author(s):  
HoangDinh Huynh ◽  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Ex Vivo ◽  
Bone Marrow Stroma ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Marrow Stroma ◽  
Igf Signaling

Abstract Abstract 1604 Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). The role of IGFBP2 in HSCs and cancer is very intriguing. IGFBP2 can bind to insulin-like growth factor (IGF) ligands and displays IGF-dependent growth inhibitory effects on many cell types. On the other hand, IGFBP2 is capable of stimulating growth of certain cancer cells, and is overexpressed in many cancer patients and its expression is correlated with cancer progression. Here we sought to study the role of IGFBP2 in regulation of the activity of normal HSCs. We showed that IGFBP2 was expressed in differentiated hematopoietic cells and bone marrow stroma but not in HSCs. Consistent with its gene expression pattern, IGFBP2-/- HSCs had similar repopulation activity as their wild-type counterparts. By contrast, when we transplanted HSCs into IGFBP2-/- or wild-type recipient mice, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-/- recipients, suggesting that the environmental IGFBP2 positively supports HSC activity. Further co-culture of HSCs with IGFBP2-/- or wild-type bone marrow stromal cells indicated that IGFBP2 produced by bone marrow stroma indeed supports HSC expansion. Consistently, HSCs in IGFBP2-/- mice showed decreased frequency and cell cycling, and had upregulated expression of cell cycle inhibitors of p21, p16, and p19. To determine whether IGFBP2's effect on HSCs depends on IGF signaling, we compared the repopulation of donor cells deficient for the IGF type I receptor in wild-type and IGFBP2-/- recipients. These HSCs that are defective in IGF signaling still have decreased repopulation in IGFBP2-/- recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF signaling. To identify the functional domain of IGFBP2 in regulation of HSC activity, we constructed IGFBP2 with mutated RGD domain or deleted c-terminus and used the mutant IGFBP2 proteins in ex vivo culture of HSCs. We found that the c-terminus of IGFBP2 is essential to support HSC activity. We are currently in the process of identifying the potential receptor of IGFBP2 on HSCs. In summary, we found that IGFBP2 supports the cycling of normal HSCs, and this effect is independent of IGF signaling. Our study is important in revealing the relationship among environmental cues and cell fates of stem cells and opens up a new avenue in investigation of the roles of IGFBP2 in stem cells and cancer. Disclosures: No relevant conflicts of interest to declare.

Effect of mutation of a calmodulin binding site on Ca2+ regulation of inositol trisphosphate receptors

2001 ◽  
Vol 360 (2) ◽  
pp. 395-400 ◽  
Author(s):  
Xianchao ZHANG ◽  
Suresh K. JOSEPH
Keyword(s):  
Splice Variants ◽  
Binding Motif ◽  
Type I ◽  
Channel Activity ◽  
Wild Type ◽  
Binding Assays ◽  
Ip3 Receptor ◽  
Calmodulin Binding ◽  
Mutant Receptors

Several studies have shown that calmodulin (CaM) modulates d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel activity and ligand binding to IP3Rs. It has been proposed that CaM may act as the Ca2+ sensor for mediating Ca2+ inhibition of IP3R channel activity. However, the functional role of CaM binding sites and the mechanism by which CaM regulates IP3R activities remains unclear. Tryptophan at position 1577 of type I IP3R has been shown to be part of a motif that is responsible for CaM binding to IP3Rs and we have mutated this residue to alanine in the long (neuronal) and short (peripheral) SII splice variants of the type I IP3R. CaM–Sepharose binding assays using COS-7 cell lysates confirmed that the W1577A mutant in both splice variants completely eliminated CaM binding. Functional measurements of IP3-mediated 45Ca2+ fluxes indicated that there was no change in the IP3 sensitivity of the channel induced by the W1577A mutation. Such measurements also indicated that the W1577A mutants of both splice variants have a dependence on external [Ca2+] that was indistinguishable from the corresponding wild-types. Although subtle differences in the Ca2+ and CaM sensitivity of [3H]IP3 binding were noted between wild-type and mutant receptors, our data suggest that the CaM binding motif involving the W1577A locus does not play a role in Ca2+ regulation of IP3R channel activity.

scholarly journals Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

2007 ◽  
Vol 404 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Gary Flom ◽  
Robert H. Behal ◽  
Luke Rosen ◽  
Douglas G. Cole ◽  
Jill L. Johnson
Keyword(s):  
Current Model ◽  
Heat Shock Protein 90 ◽  
C Terminus ◽  
Client Proteins ◽  
Definition Of ◽  
Necessary And Sufficient ◽  
In Vitro Interaction

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

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