Surveying the serologic proteome in a tissue‐specific kras(G12D) knockin mouse model of pancreatic cancer

Ludwig, Michael; Kojima, Kensuke; Bowersock, Gregory J.; Chen, Dongquan; Jhala, Nirag; Buchsbaum, Donald J.; Grizzle, William E.; Klug, Christopher A.; Mobley, James A.

doi:10.1002/pmic.201500133

Cited by 41 publications

(34 citation statements)

References 72 publications

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“…Mass spectrometry and data analysis was conducted as described in (37) using an Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher Scientific). Duplicate samples were run and data represents pooled results.…”

Section: Methodsmentioning

confidence: 99%

Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases

et al. 2016

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CRISPR–Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR–Cas system that encodes the Cas10–Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10–Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10–Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10–Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR–Cas immunity and central nucleic acid metabolism.

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Section: Methodsmentioning

confidence: 99%

Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases

et al. 2016

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“…Peptide extracts were reconstituted in 0.1% formic acid/ddH 2 O at 0.1 µg/µL. Electrospray ionization tandem mass spectrometry was carried out, and the data were processed, searched, filtered, grouped, and quantified, as previously reported in detail (Ludwig et al 2016). Mass spectrometry was performed by the UAB Cancer Center Mass Spectrometry and Proteomics Shared Facility.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Nasef

Muffly

Beckman

et al. 2019

RNA

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CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg 2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.

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“…All data were collected in collision-induced dissociation mode. The instrument configuration during data collection followed previous publication [18–20]. …”

Section: Methodsmentioning

confidence: 99%

Comparative proteomic analysis of three Chinese hamster ovary (CHO) host cells

Sun

et al. 2017

Biochemical Engineering Journal

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Chinese hamster ovary (CHO)1 cells have been widely used to express heterologous genes and produce therapeutic proteins in biopharmaceutical industry. Different CHO host cells have distinct cell growth rates and protein expression characteristics. In this study, the expression of about 1,307 host proteins in three sublines, i.e. CHO K1, CHO S and CHO/dihydrofolate reductase (dhfr)−, were investigated and compared using proteomic analysis. The proteins involved in cell growth, glycolysis, tricarboxylic acid cycle, transcription, translation and glycosylation were quantitated using Liquid chromatography tandem-mass spectrometry (LC-MS/MS). The key host cell proteins that regulate the kinetics of cell growth and the magnitude of protein expression levels were identified. Furthermore, several rational cell engineering strategies on how to combine the desired features of fast cell growth and efficient production of therapeutic proteins into one new super CHO host cell have been proposed.

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Surveying the serologic proteome in a tissue‐specific kras(G12D) knockin mouse model of pancreatic cancer

Cited by 41 publications

References 72 publications

Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases

Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Comparative proteomic analysis of three Chinese hamster ovary (CHO) host cells

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