Purification and Refolding to Amyloid Fibrils of (His)&lt;sub&gt;6&lt;/sub&gt;-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in&lt;em&gt; E. coli&lt;/em&gt;

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidić, Jasmina

doi:10.3791/53432

Cited by 3 publications

(2 citation statements)

References 23 publications

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“…Primary human keratinocytes were cultured in Keratinocyte Serum Free Media supplemented with Epidermal Growth Factor 1-53 and Bovine Pituitary Extract (Life Technologies; 17005042). For generating organotypic skin cultures, ~500 K control or knockdown cells were seeded on devitalized human dermis and raised to an air/liquid interface in order to induce differentiation and stratification over the indicated number of days with culture changes every 2 days 64 .…”

Section: Methodsmentioning

confidence: 99%

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

et al. 2020

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How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

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Section: Methodsmentioning

confidence: 99%

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

et al. 2020

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“…For a His-tagged protein, load the supernatant onto an equilibrated, nickel-charged, immobilized metal affinity chromatography (IMAC) column using a fast protein liquid chromatography (FPLC) system (see the table of materials) 52 .…”

Section: Protocolmentioning

confidence: 99%

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Yanez-Orozco

Adariani

et al. 2017

JoVE

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A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all “dimensions” of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

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Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus

et al. 2019

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The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrPC). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrPSc), levels of mature endogenous Sho become reduced. To study the regulatory effect of the proteostatic network on Sho expression, we investigated the action of lactacystin, MG132, NH4Cl, and 3-methyladenine (3-MA) in two cell culture models. In primary mixed neuronal and glial cell cultures (MNGCs) from transgenic mice expressing wild-type Sho from the PrP gene promoter (Tg.Sprn mice), lactacystin- and MG132-mediated inhibition of proteasomal activity shifted the repertoire of Sho species towards unglycosylated forms appearing in the nuclei; conversely, the autophagic modulators NH4Cl and 3-MA did not affect Sho or PrPC glycosylation patterns. Mouse N2a neuroblastoma cells expressing Sho under control of a housekeeping gene promoter treated with MG132 or lactacystin also showed increased nuclear localization of unglycosylated Sho. As two proteasomal inhibitors tested in two cell paradigms caused redirection of Sho to nuclei at the expense of processing through the secretory pathway, our findings define a balanced shift in subcellular localization that thereby differs from the decreases in net Sho species seen in prion-infected brains. Our data are indicative of a physiological pathway to access Sho functions in the nucleus under conditions of impaired proteasomal activity. We also infer that these conditions would comprise a context wherein Sho’s N-terminal nucleic acid–binding RGG repeat region is brought into play.Electronic supplementary materialThe online version of this article (10.1007/s12035-019-1623-1) contains supplementary material, which is available to authorized users.

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Purification and Refolding to Amyloid Fibrils of (His)<sub>6</sub>-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in<em> E. coli</em>

Cited by 3 publications

References 23 publications

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus

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