Uncovering the cellular capacity for intensive and specific feedback self-control of the argonautes and MicroRNA targeting activity

Wang, Degeng; Wang, Tingzeng; Gill, Audrey; Hilliard, Terrell; Chen, Fengqian; Karamyshev, Andrey L.; Zhang, Fangyuan

doi:10.1093/nar/gkaa209

Cited by 14 publications

(24 citation statements)

References 65 publications

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“…We downloaded the PhosphoSitePlus database (www.phosphosite.org) dataset and counted the number of unique experimentally verified substrates for each human protein kinases, revealing that human AKT1 has 215 unique substrates (45). As many other cell biology parameters (46)(47)(48)(49), the substrate count (K) of the human kinome follows the so-called scalefree distribution, i.e., P (k) ∝ K −α or log 2 (P (k) ) ∝ -α*log 2 (K), with P (K) as the portion of protein kinases with K unique substrates and α as a positive constant. This is illustrated in the linear relationship in a log-log plot (log 2 (P (k) vs. log 2 (K)) in Figure 3.…”

Section: Generation and Expression Of Mutant Akt1 Kinasementioning

confidence: 99%

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

Chen

Liu

Hilliard

et al. 2020

Preprint

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The human kinome contains >500 protein kinases, and regulates up to 30% of all human proteins. Kinase study is currently hindered by a lack of in vivo analysis approaches, mainly due to two factors: our inability to distinguish the kinase reaction of interest from those of other members of the kinome in live cells and the cell impermeability of the ATP analogs. Herein, we aimed to overcome this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the Shokat method, was used as our initial prototype. Briefly, following the Shokat method, enlargement of the ATP binding pocket was performed by mutating the gate-keeper Methionine residue to a Glycine, prompting the mutant AKT1 to preferentially use the bulky ATP analog N 6 -Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The LPC nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. We demonstrated that the mutant, but not the wild type, AKT1 was able to use the delivered A*TPγS for autophosphorylation as well as phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

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Section: Generation and Expression Of Mutant Akt1 Kinasementioning

confidence: 99%

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

Chen

Liu

Hilliard

et al. 2020

Preprint

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“…HCT116 human colon cancer cells, originally obtained from Dr. Brattain's lab (30)(31)(32)(33), were cultured in the McCoy's 5A medium (Invitrogen, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, USA). Cells were cultivated at 37 °C and 5 % CO 2 in an incubator.…”

Section: Cell Culturementioning

confidence: 99%

Enablingin vivoAnalysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype

Chen

Liu

Hilliard

et al. 2020

Preprint

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AbstractMany experimental protocols have to be carried out in vitro due to a lack of cell-permeable analysis probes. For instance, the cellular signaling moderator RAS proteins alternate between the active GTP-binding and the inactive GDP-binding states. Though many GTP analogs can serve as probes for RAS activity analysis, their cell impermeability renders in vivo analysis impossible. On the other hand, the lipid/calcium/phosphate (LCP) nanoparticle has enabled efficient intracellular delivery of a nucleotide analog as a chemotherapy agent. Thus, using RAS analysis and LCP nanoparticle as the prototype, we tackled the cell-impermeability issue via nanoparticle-mediated intracellular delivery of the analysis probe. Briefly, BODIPY-FT-GTP-γ-S, a GTP analog that becomes fluorescent only upon protein binding, was chosen as the analysis probe, so that GTP binding can be quantified by fluorescent activity. BODIPY-FT-GTP-γ-S-loaded LCP-nanoparticle was synthesized for efficient intracellular BODIPY-FT-GTP-γ-S delivery. Binding of the delivered BODIPY-FT-GTP-γ-S to the RAS proteins were consistent with previously reported observations; the RAS GTP binding activity was reduced in serum-starved cells; and a transient activation peak of the binding activity was observed upon subsequent serum reactivation of the cells. In a word, nanoparticle-mediated probe delivery enabled an in vivo RAS analysis method. The approach should be applicable to a wide variety of analysis protocols.

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“…MiRNAs are generated by stepwise cleavage via double-stranded ribonuclease III enzymes DROSHA and DICER1 and loaded onto Argonaute (AGO) proteins to pair with target sequences on mRNAs 20 . The miRNA-mRNA pairing triggers casein kinase 1α (CK1α)-induced phosphorylation and dissociation of AGO2 from the active complex, whereas the ANKRD52-PPP6C phosphatase complex dephosphorylates AGO2 to restore its miRNA loading activity 21 , 22 . Recurrent mutations in these core miRNA machinery components are identified in many cancer types including neuroblastoma, Wilms tumor, ovarian cancer, and melanoma 23 – 28 .…”

Section: Introductionmentioning

confidence: 99%

Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

et al. 2021

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Cancer cells acquire genetic heterogeneity to escape from immune surveillance during tumor evolution, but a systematic approach to distinguish driver from passenger mutations is lacking. Here we investigate the impact of different immune pressure on tumor clonal dynamics and immune evasion mechanism, by combining massive parallel sequencing of immune edited tumors and CRISPR library screens in syngeneic mouse tumor model and co-culture system. We find that the core microRNA (miRNA) biogenesis and targeting machinery maintains the sensitivity of cancer cells to PD-1-independent T cell-mediated cytotoxicity. Genetic inactivation of the machinery or re-introduction of ANKRD52 frequent patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1). Expression of each miRNA machinery component strongly correlates with intratumoral T cell infiltration in nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway can be exploited by cancer cells to escape from T cell-mediated elimination and immunotherapy.

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Uncovering the cellular capacity for intensive and specific feedback self-control of the argonautes and MicroRNA targeting activity

Cited by 14 publications

References 65 publications

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

A Chemical-genetics and Nanoparticle Enabled Approach forin vivoProtein Kinase Analysis

Enablingin vivoAnalysis Via Nanoparticle-mediated Intracellular Assay Probe Delivery: Using RAS as the Prototype

Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

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