Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.

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Miga-mediated endoplasmic reticulum–mitochondria contact sites regulate neuronal homeostasis

eLife ◽

10.7554/elife.56584 ◽

2020 ◽

Vol 9 ◽

Author(s):

Lingna Xu ◽

Xi Wang ◽

Jia Zhou ◽

Yunyi Qiu ◽

Weina Shang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Mitochondrial Dynamics ◽

Molecular Organization ◽

Lipid Transport ◽

Cellular Processes ◽

Contact Sites ◽

Neuronal Homeostasis ◽

Higher Eukaryotes ◽

Lateral Sclerosis

Endoplasmic reticulum (ER)–mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.

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ROS as a Novel Indicator to Predict Anticancer Drug Efficacy

10.21203/rs.2.11911/v2 ◽

2019 ◽

Author(s):

Tarek Zaidieh ◽

James Smith ◽

Karen Ball ◽

Qian An

Keyword(s):

Cancer Cells ◽

Copy Number ◽

Drug Sensitivity ◽

Drug Efficacy ◽

Ros Generation ◽

Targeting Therapy ◽

Mitochondrial Dna Copy Number ◽

Conventional Drug ◽

Apoptosis Pathways ◽

Mitochondria Targeting

Abstract Background Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. Generally, cancer cells with mitochondrial genetic abnormalities (copy number change and mutations) have escalated ROS levels compared to normal cells. Since high levels of ROS can trigger apoptosis, treating cancer cells with low doses of mitochondria-targeting / ROS-stimulating agents may offer cancer-specific therapy. This study aimed to investigate how baseline ROS levels might influence cancer cells’ response to ROS-stimulating therapy. Methods Four cancer and one normal cell lines were treated with a conventional drug (cisplatin) and a mitochondria-targeting agent (dequalinium chloride hydrate) separately and jointly. Cell viability was assessed and drug combination synergisms were indicated by the combination index (CI). Mitochondrial DNA copy number (MtDNAcn), ROS and mitochondrial membrane potential (MMP) were measured, and the relative expression levels of the genes and proteins involved in ROS-mediated apoptosis pathways were also investigated. Results Our data showed a correlation between the baseline ROS level, mtDNAcn and drug sensitivity in the tested cells. Synergistic effect of both drugs was also observed with ROS being the key contributor in cell death. Conclusions Our findings suggest that mitochondria-targeting therapy could be more effective compared to conventional treatments. In addition, cancer cells with low levels of ROS may be more sensitive to the treatment, while cells with high levels of ROS may be more resistant. This study provides an insight into understanding the influence of intracellular ROS on drug sensitivity, and may lead to the development of new therapeutic strategies to improve efficacy of anticancer therapy.

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Changes in numbers of prolactin receptors during the cell cycle of Nb2 cells

Journal of Endocrinology ◽

10.1677/joe.0.159r001 ◽

1998 ◽

Vol 159 (1) ◽

pp. R1-R4

Author(s):

IG Camarillo ◽

JA Rillema

Keyword(s):

Cell Cycle ◽

Specific Binding ◽

Prolactin Receptors ◽

Initial Value ◽

Cellular Processes ◽

Nb2 Cells ◽

Signalling Molecule ◽

Lactogenic Hormones ◽

T Lymphoma ◽

Stimulation Of

Lactogenic hormones including prolactin (PRL) have mitogenic effects on Nb2 cells, a pre-T lymphoma cell line. Previous studies have characterized the PRL stimulation of cellular processes such as RNA/DNA synthesis, signalling molecule activation, and the expression of specific genes. The data presented here explores the fluctuations in plasma membrane PRL receptor (PRLR) number that occur in the Nb2 cells during the course of a 24 h cell cycle. PRLR abundance was determined by measuring specific binding of [I125] oPRL to G1 arrested-intact Nb2 cells in which the cell cycle was initiated by addition of nonradioactive oPRL. Preliminary studies revealed that 1 ng/ml oPRL was the minimum PRL concentration that causes a maximal stimulation of mitogenesis, without interfering with [I125] oPRL binding measurements. Subsequent experiments revealed that upon cell cycle initiation of G1 arrested Nb2 cells with 1 ng/ml oPRL, PRLR number remained constant for the initial 6 h. After 8 h PRLR numbers decreased and at 12 h, the PRLR number was less than 25% of the initial value. After 12 hr, PRLR numbers increased and reach initial values by 18 hr. These studies show that the expression of cell surface PRL receptors is modulated in a sequential fashion during the cell cycle of Nb2 cells.

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Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

International Journal of Molecular Sciences ◽

10.3390/ijms21145004 ◽

2020 ◽

Vol 21 (14) ◽

pp. 5004

Author(s):

Ekaterina O. Serebrovskaya ◽

Nadezda M. Podvalnaya ◽

Varvara V. Dudenkova ◽

Anna S. Efremova ◽

Nadya G. Gurskaya ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Mammalian Cells ◽

Fluorescent Proteins ◽

Fluorescent Sensor ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Post Translational Modification ◽

Cellular Processes ◽

Hydrogen Peroxide Treatment ◽

Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

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Functional genetic encoding of sulfotyrosine in mammalian cells

Nature Communications ◽

10.1038/s41467-020-18629-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Xinyuan He ◽

Yan Chen ◽

Daisy Guiza Beltran ◽

Maia Kelly ◽

Bin Ma ◽

...

Keyword(s):

Mammalian Cells ◽

Biochemical Characterization ◽

Trna Synthetase ◽

Functional Verification ◽

Cellular Processes ◽

Genetic Encoding ◽

Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

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Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives

Biomolecules ◽

10.3390/biom10111535 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1535

Author(s):

Renata Tisi ◽

Vadim Gaponenko ◽

Marco Vanoni ◽

Elena Sacco

Keyword(s):

Natural Products ◽

Mammalian Cells ◽

Developmental Disorders ◽

Health Gain ◽

Point Mutations ◽

Ras Signaling ◽

Cellular Processes ◽

Oncogenic Ras ◽

Ras Genes ◽

Critical Issues

RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.

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56 Sirtinol Treatment Influences Preimplatation Development of Porcine Embryos via Regulation of Autophagy and Apoptosis

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab56 ◽

2018 ◽

Vol 30 (1) ◽

pp. 167

Author(s):

M. G. Kim ◽

S. T. Shin ◽

H. D. Shin ◽

H. T. Lee

Keyword(s):

Technology Development ◽

Development Program ◽

Treated Group ◽

Blastocyst Stage ◽

Class Iii ◽

Protein Levels ◽

Cellular Processes ◽

Maternal Mrna ◽

Apoptosis Pathways

Sirtuin (Sirt), nicotinamide adenine dinucleotide dependent class III histone deacetylase, plays an important role in cellular processes including DNA repair, apoptosis, cell cycle, aging, and determining lifespan. In previous studies, levels of Sirt1 to Sirt3 mRNA were detected in porcine embryos for the first time and levels are lower in blastocysts relative to matured oocytes. In addition, mitochondrial dysfunction and hyperglycemia increases LC3 protein levels and apoptosis in porcine parthenotic embryos and modulation of autophagy also influences apoptosis, mitochondrial contents, abnormal autophagosome formation, and maternal mRNA degradation. However, Sirt-mediated mechanisms have not been examined in in vitro-produced embryos of pig. Therefore, we investigated the relationship between Sirt inhibition and autophagy/mitophagy in porcine pre-implantation embryos. After IVF, embryos were cultured in NCSU-23 media in the presence and absence of 100 μM sirtinol (Sirt inhibitor) until the expended blastocyst stage. As a result, there were no significant differences between the rate of cleavage in control (69.22 ± 1.29) and treated groups (72.66 ± 1.08). However, embryos treated with sirtinol had significantly decreased developmental rates to the morula as well as blastocyst stages. Especially, expanded blastocysts (9.90 ± 1.56 v. 2.92 ± 0.94%) were barely observed in sirtinol-treated group. In the levels of Sirt transcripts, Sirt2 mRNA was significantly lower in sirtinol-treated blastocysts compared with controls (P < 0.05), but the levels of Sirt1 and Sirt3 mRNA were similar in both groups. In addition, we found that sirtinol treatment induced autophagy by increasing the expressions of LC3, Beclin1, and ATG5 in blastocysts. Furthermore, we observed that the abundance of mitochondria stained with mitotracker was lower in sirtinol-treated blastocysts than that of control. Finally, we found that sirtinol treatment resulted in a higher total apoptotic index (6.88 ± 0.84) compared with the control (12.84 ± 0.99) in blastocysts. In summary, our findings in this study demonstrated that Sirt inhibition by sirtinol led to lower levels of Sirt2 transcript in blastocysts, reduced developmental capability and embryo quality with regulation of ATGs, LC3 proteins, apoptosis-related genes, and mitochondrial abundance. Therefore, these results suggest that Sirt2 may play an important role in the pre-implantation development of porcine embryos and their quality through the regulation of autophagy/mitophagy and apoptosis pathways. This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.

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Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

Biomolecules ◽

10.3390/biom10101403 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1403

Author(s):

Maryia Karpiyevich ◽

Katerina Artavanis-Tsakonas

Keyword(s):

Mammalian Cells ◽

Dynamic Balance ◽

Fine Tuning ◽

Protozoan Parasites ◽

Cellular Functions ◽

Parasitic Protozoa ◽

Distinctive Features ◽

Cellular Processes ◽

Wide Range ◽

Enzymatic Machinery

Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.

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Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0412 ◽

2017 ◽

Vol 28 (13) ◽

pp. 1768-1781 ◽

Cited By ~ 13

Author(s):

Alejandra Valdivia ◽

Silvia M. Goicoechea ◽

Sahezeel Awadia ◽

Ashtyn Zinn ◽

Rafael Garcia-Mata

Keyword(s):

Cell Migration ◽

Mammalian Cells ◽

Dorsal Surface ◽

Receptor Internalization ◽

Dependent Manner ◽

Cellular Functions ◽

Unique Type ◽

Exchange Factor ◽

Cellular Processes

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation.

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