[20] Solubilization of protein aggregates

Marston, Fiona A. O.; Hartley, Donna F.

doi:10.1016/0076-6879(90)82022-t

Cited by 118 publications

(59 citation statements)

References 29 publications

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“…3F). This possibility was confirmed by treatment of cytoskeletal preparations with 8 M urea to dissociate neurofilament complexes (Marston and Hartley, 1990), which eliminated RMO-24 immunoreactivity within the stack and increased immunoreactivity associated with the anticipated migratory position for extensively phosphorylated NF-H. Notably, some SMI-31 and SMI-34 reactivity was observed within this aggregate (Fig.…”

Section: Ck1 Mediates the Major Retardation Of Nf-h Migration On Sds mentioning

confidence: 67%

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

Lee

Pant²,

Shea

2014

Journal of Cell Science

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C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogenactivated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3b (GSK3b). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li + , D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3b, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3b mediated the neurofilamentneurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/ 2) was found to downregulate GSK3b, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilamentneurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3b, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3b.

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Section: Ck1 Mediates the Major Retardation Of Nf-h Migration On Sds mentioning

confidence: 67%

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

Lee

Pant²,

Shea

2014

Journal of Cell Science

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“…Inclusion bodies were purified as follows (Marston & Hartley, 1990). Cell pellets were sonicated on ice in 50 mM Tris.HCI, pH 8.7, 1 mM EDTA, and spun at 35,000 x g for 20 min.…”

Section: Purification Of Recombinant Proteinmentioning

confidence: 99%

Formation of a native‐like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor

1994

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In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-511 plays a key role. We have investigated a recombinant analog of [30-511 using 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51lAla, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, ,,,, consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-511, named PaPo, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-511 and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of The transient nature of most protein folding intermediates precludes a detailed structural study of these intermediates (for review, see Matthews, 1993). Conveniently, however, intermediates in the folding pathway of bovine pancreatic trypsin inhibitor are distinguished covalently by the disulfide bonds that they contain (Creighton, 1977;Creighton & Goldenberg, 1984;Weissman & Kim, 1991, 1992a. The covalent nature of these intermediates (Fig. 1) allows them to be trapped readily and characterized in structural detail. Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; COSY, correlation spectroscopy; Gdn. HCI, guanidine hydrochloride; HMQC, heteronuclear multiple quantum coherence; HSMQC, heteronuclear single-multiple quantum coherence; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; ppm, parts per million; TOCSY, total correlation spectroscopy; 2D-NMR, 2-dimensional NMR.94143-0448.

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“…The insoluble material was pelleted and solubilized in a solution consisting of 4 M guanidinium hydrochloride, 100 mM Tris, and 100 mM DTT (final pH, 8.2) by 30 min of shaking at room temperature. The protein solution was brought to pH 3.0 by the addition of 1 M hydrochloric acid and dialyzed overnight in 100 mM acetic acid with 1 mM DTT (25). The protein was stored frozen in 100-l aliquots at Ϫ80°C until needed.…”

Section: Methodsmentioning

confidence: 99%

SdeK, a Histidine Kinase Required for Myxococcus xanthus Development

Pollack

Singer

2001

J Bacteriol

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The sdeK gene is essential to the Myxococcus xanthus developmental process. We reported previously, based on sequence analysis (A. G. Garza, J. S. Pollack, B. Z. Harris, A. Lee, I. M. Keseler, E. F. Licking, and M. Singer, J. Bacteriol. 180:4628-4637, 1998), that SdeK appears to be a histidine kinase. In the present study, we have conducted both biochemical and genetic analyses to test the hypothesis that SdeK is a histidine kinase. An SdeK fusion protein containing an N-terminal polyhistidine tag (His-SdeK) displays the biochemical characteristics of a histidine kinase. Furthermore, histidine 286 of SdeK, the putative site of phosphorylation, is required for both in vitro and in vivo protein activity. The results of these assays have led us to conclude that SdeK is indeed a histidine kinase. The developmental phenotype of a ⌬sdeK1 strain could not be rescued by codevelopment with wild-type cells, indicating that the defect is not due to the mutant's inability to produce an extracellular signal. Furthermore, the ⌬sdeK1 mutant was found to produce both A-and C-signal, based on A-factor and codevelopment assays with a csgA mutant, respectively. The expression patterns of several Tn5lacZ transcriptional fusions were examined in the ⌬sdeK1-null background, and we found that all C-signaldependent fusions assayed also required SdeK for full expression. Our results indicate that SdeK is a histidine kinase that is part of a signal transduction pathway which, in concert with the C-signal transduction pathway, controls the activation of developmental-gene expression required to progress past the aggregation stage.

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[20] Solubilization of protein aggregates

Cited by 118 publications

References 29 publications

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

Formation of a native‐like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor

SdeK, a Histidine Kinase Required for Myxococcus xanthus Development

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