Evidence map›Paper›PMID 34762224›Full record

ArticleMolecular biology reports2022

Development of a one-plasmid system to replace the endogenous protein with point mutation for post-translational modification studies.

Iqra Ishrat, Aoxing Cheng, Fazhi Yu, Jing Guo, Peng Zhang, Kaiguang Zhang, Zhenye Yang

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In one paragraph

Article in Molecular biology reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.

0numbers the graph read from it
0cells of the map it votes in
1citing papers in PubMed
0.9field-weighted citation impact, top 23% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

1 citing paper in PubMed, 5 citations in OpenAlex.

  1. Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

7 authors at 1 institution in 1 country.

Iqra IshratDepartment of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China.
Aoxing ChengDepartment of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China.
Fazhi YuHefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Jing GuoDivision of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China.
Peng ZhangHefei National Laboratory for Physical Sciences at Microscale, The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China. peng0912@ustc.edu.cn.
Kaiguang ZhangDepartment of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China. zhangkaiguang@ustc.edu.cn.
Zhenye YangDepartment of Digestive Disease, The First Affiliated Hospital of USTC, Anhui Provincial Hospital, University of Science and Technology of China, Hefei, China. zhenye@ustc.edu.cn.ORCID http://orcid.org/0000-0001-7797-5891
University of Science and Technology of China · CN

Funding

National Natural Science Foundation of China 31771498
6 · The paper itself

Abstract

backgroundPost-translational modification (PTM) is one of the major regulatory mechanism for protein activities. To understand the function of PTMs, mutants that prevent or mimic the modification are frequently utilized. The endogenous proteins are usually depleted while the point mutations are expressed. A common strategy to accomplish these tasks includes two-steps: First, a cell line stably expressing shRNA for protein depletion is generated, then an RNAi-resistance construct is introduced to express mutant. However, these steps are time- and labor-consuming. More importantly, shRNA and mutant protein are frequently expressed in different cells at different time, which significantly disturbs the conclusions.

methodsTo overcome these technical problems, we developed a lentiviral based one-plasmid system that allowed concurrent expression of shRNA and mutant protein. The puromycin-resistant gene was inserted for the selection of stable-expression cells.

resultsUsing this plasmid, we efficiently replaced the endogenous proteins with comparable levels of exogenous proteins for LDHB and PKM2, two glycolytic enzymes regulated by PTM in cancer cells. The system was also successfully exploited in evaluating the role of phosphorylation of LDHB serine 162 in multiple in vitro and in vivo assays.

conclusionThus, we have developed an efficient one-plasmid system to replace endogenous protein with point mutations for the functional study of PTM.

Indexed as

Point MutationAnimalsCarrier ProteinsCell Line, TumorHCT116 CellsHeLa CellsHep G2 CellsHumansIsoenzymesL-Lactate DehydrogenaseMaleMembrane ProteinsMicePhosphorylationPlasmidsProtein Processing, Post-TranslationalCarrier ProteinsIsoenzymeslactate dehydrogenase 1L-Lactate DehydrogenaseMembrane ProteinsRNA, Small InterferingSerineThyroid Hormone-Binding ProteinsThyroid HormonesKnockdownpGIPZ vectorPoint mutationPTMStable expression

Identifiers

PMID34762224
OpenAlexW3213973563

What OpenQuestion holds

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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.