scholarly journals A kinesin-1 variant reveals motor-induced microtubule damage in cells

2021 ◽  
Author(s):  
Breane G Budaitis ◽  
Somayesadat Badieyan ◽  
Yang Yue ◽  
T Lynne Blasius ◽  
Dana N Reinemann ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Human Disease ◽  
Cultured Cells ◽  
Wild Type ◽  
Post Translational Modification ◽  
Lattice Damage ◽  
Microtubule Growth ◽  
Type Motor ◽  
In Cells

Kinesins drive the transport of cellular cargoes as they walk along microtubule tracks, however, recent work has suggested that the physical act of kinesins walking along microtubules can stress the microtubule lattice. Here, we describe a kinesin-1 KIF5C mutant with an increased ability to generate defects in the microtubule lattice as compared to the wild-type motor. Expression of the mutant motor in cultured cells resulted in microtubule breakage and fragmentation, suggesting that kinesin-1 variants with increased damage activity would have been selected against during evolution. The increased ability to damage microtubules is not due to the altered motility properties of the mutant motor as expression of the kinesin-3 motor KIF1A, which has similar single-motor motility properties, also caused increased microtubule pausing, bending, and buckling but not breakage. In cells, motor-induced microtubule breakage could not be prevented by increased α-tubulin K40 acetylation, a post-translational modification known to increase microtubule flexibility. In vitro, lattice damage induced by wild-type KIF5C was repaired by soluble tubulin and resulted in increased rescues and microtubule growth whereas lattice damage induced by the KIF5C mutant resulted in larger repair sites that made the microtubule vulnerable to breakage and fragmentation when under mechanical stress. These results demonstrate that kinesin-1 motility causes defects in and damage to the microtubule lattice in cells. While cells have the capacity to repair lattice damage, conditions that exceed this capacity result in microtubule breakage and fragmentation and may contribute to human disease.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

scholarly journals Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo

2002 ◽  
Vol 76 (2) ◽  
pp. 717-729 ◽  
Author(s):  
Maryam Ahmed ◽  
Martin Lock ◽  
Cathie G. Miller ◽  
Nigel W. Fraser
Keyword(s):  
Herpes Simplex ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Exon 1 ◽  
Induced Apoptosis ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Recent studies have suggested that the latency-associated transcript (LAT) region of herpes simplex virus type 1 (HSV-1) is effective at blocking virus-induced apoptosis both in vitro and in the trigeminal ganglia of acutely infected rabbits (Inman et al., J. Virol. 75:3636–3646, 2001; Perng et al., Science 287:1500–1503, 2000). By transfecting cells with a construct expressing the Pst-Mlu segment of the LAT, encompassing the LAT exon 1, the stable 2.0-kb intron, and 5′ part of exon 2, we confirmed that this region was able to diminish the onset of programmed cell death initiated by anti-Fas and camptothecin treatment. In addition, caspase 8-induced apoptosis was specifically inhibited in cells expressing the Pst-Mlu LAT fragment. To further delineate the minimal region of LAT that is necessary for this antiapoptotic function, LAT mutants were used in our cotransfection assays. In HeLa cells, the plasmids lacking exon sequences were the least effective at blocking apoptosis. However, similar to previous work (Inman et al., op. cit.), our data also indicated that the 5′ end of the stable 2.0-kb LAT intron appeared to contribute to the promotion of cell survival. Furthermore, cells productively infected with the 17N/H LAT mutant virus, a virus deleted in the LAT promoter, exon 1, and about half of the intron, exhibited a greater degree of DNA fragmentation than cells infected with wild-type HSV-1. These data support the finding that the exon 1 and 2.0-kb intron region of the LAT transcription unit display an antiapoptotic function both in transfected cells and in the context of the virus infection in vitro. In trigeminal ganglia of mice acutely infected with the wild-type virus, 17, and 17ΔSty, a virus lacking most of exon 1, apoptosis was not detected in cells that were positive for virus particles. However, dual staining was observed in cells from mice infected with 17N/H virus, indicating that the LAT antiapoptotic function demonstrated in cells transfected by LAT-expressing constructs may also play a role in protecting cells from virus-induced apoptosis during acute viral infection in vivo.

scholarly journals The UL47 gene of avian infectious laryngotracheitis virus is not essential for in vitro replication but is relevant for virulence in chickens

2007 ◽  
Vol 88 (3) ◽  
pp. 732-742 ◽  
Author(s):  
Dorothee Helferich ◽  
Jutta Veits ◽  
Jens P. Teifke ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Deletion Mutant ◽  
Cultured Cells ◽  
Functional Characterization ◽  
Wild Type Virus ◽  
Wild Type ◽  
Infectious Laryngotracheitis Virus ◽  
Live Virus ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus

The genome of infectious laryngotracheitis virus (ILTV) exhibits several differences from those of other avian and mammalian alphaherpesviruses. One of them is the translocation of the conserved UL47 gene from the unique long (UL) to the unique short (US) genome region, where UL47 is inserted upstream of the US4 gene homologue. As in other alphaherpesviruses, UL47 encodes a major tegument protein of ILTV particles, whereas the US4 gene product is a non-structural glycoprotein, gG, which is secreted from infected cells. For functional characterization, an ILTV recombinant was isolated in which US4 together with the 3′-terminal part of UL47 was replaced by a reporter gene cassette encoding green fluorescent protein. From this virus, UL47 and US4 single-gene deletion mutants without foreign sequences were derived and virus revertants were also generated. In vitro studies revealed that both genes were non-essential for ILTV replication in cultured cells. Whereas US4-negative ILTV exhibited no detectable growth defects, maximum virus titres of the double deletion mutant and of UL47-negative ILTV were reduced about 10-fold compared with those of wild-type virus and rescued virus. Experimental infection of chickens demonstrated that UL47-negative ILTV was significantly attenuated in vivo and was shed in reduced amounts, whereas wild-type and rescued viruses caused severe disease and high mortality rates. As all immunized animals were protected against subsequent challenge infection with virulent ILTV, the UL47 deletion mutant might be suitable as a live-virus vaccine.

scholarly journals O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

2020 ◽  
pp. jbc.RA120.016116
Author(s):  
Ravin Seepersaud ◽  
Alexander C. Anderson ◽  
Barbara A. Bensing ◽  
Biswa P Choudhury ◽  
Anthony J. Clarke ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Regulatory Mechanism ◽  
Human Platelets ◽  
Bacterial Virulence ◽  
Wild Type ◽  
Post Translational Modification ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
Platelet Binding

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec (aSec) system. While all aSec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin GspB. Since these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues, and that O-acetylation prevented Glc deposition. Whereas streptococci expressing non-acetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to wild-type levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, since O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

scholarly journals Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Kazunori Sano ◽  
Yasushi Iwasaki ◽  
Yuta Yamashita ◽  
Keiichi Irie ◽  
Masato Hosokawa ◽  
...  
Keyword(s):  
Lewy Body ◽  
Cultured Cells ◽  
Lewy Body Dementia ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Central Feature ◽  
Aggregate Formation ◽  
Wild Type ◽  
Brain Involvement

AbstractSerine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Shejuan An ◽  
Jeannie M. Camarillo ◽  
Tina Yi-Ting Huang ◽  
Daphne Li ◽  
Juliette A. Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

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