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1.
Cell Stem Cell ; 28(11): 1950-1965.e6, 2021 11 04.
Article in English | MEDLINE | ID: mdl-34388375

ABSTRACT

Maintaining proteostasis is key to resisting stress and promoting healthy aging. Proteostasis is necessary to preserve stem cell function, but little is known about the mechanisms that regulate proteostasis during stress in stem cells, and whether disruptions of proteostasis contribute to stem cell aging is largely unexplored. We determined that ex-vivo-cultured mouse and human hematopoietic stem cells (HSCs) rapidly increase protein synthesis. This challenge to HSC proteostasis was associated with nuclear accumulation of Hsf1, and deletion of Hsf1 impaired HSC maintenance ex vivo. Strikingly, supplementing cultures with small molecules that enhance Hsf1 activation partially suppressed protein synthesis, rebalanced proteostasis, and supported retention of HSC serial reconstituting activity. Although Hsf1 was dispensable for young adult HSCs in vivo, Hsf1 deficiency increased protein synthesis and impaired the reconstituting activity of middle-aged HSCs. Hsf1 thus promotes proteostasis and the regenerative activity of HSCs in response to culture stress and aging.


Subject(s)
Hematopoietic Stem Cells , Proteostasis , Aging , Animals , Cellular Senescence , Mice , Transcription Factors
2.
Cancers (Basel) ; 12(6)2020 Jun 10.
Article in English | MEDLINE | ID: mdl-32531927

ABSTRACT

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

3.
Cell Rep ; 30(1): 69-80.e6, 2020 01 07.
Article in English | MEDLINE | ID: mdl-31914399

ABSTRACT

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.


Subject(s)
Cell Self Renewal , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Proteome/metabolism , Animals , Mice, Inbred C57BL , Myeloid Progenitor Cells/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis , Protein Stability , Protein Unfolding , Proto-Oncogene Proteins c-myc/metabolism , RNA Editing/genetics , RNA, Transfer/genetics , Ubiquitination
4.
FASEB J ; 33(6): 6778-6788, 2019 06.
Article in English | MEDLINE | ID: mdl-30807703

ABSTRACT

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.


Subject(s)
DNA Breaks, Double-Stranded/radiation effects , DNA End-Joining Repair/physiology , DNA Ligase ATP/metabolism , DNA Repair/physiology , Genomic Instability , Human Embryonic Stem Cells/physiology , Poly-ADP-Ribose Binding Proteins/metabolism , Cells, Cultured , DNA End-Joining Repair/radiation effects , DNA Ligase ATP/genetics , DNA Repair/radiation effects , Homologous Recombination , Human Embryonic Stem Cells/cytology , Humans , Poly-ADP-Ribose Binding Proteins/genetics
5.
Cells ; 8(1)2019 01 15.
Article in English | MEDLINE | ID: mdl-30650618

ABSTRACT

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.


Subject(s)
Dystrophin/deficiency , Genomic Instability , Induced Pluripotent Stem Cells/metabolism , Muscular Dystrophy, Duchenne/genetics , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type I/metabolism , Cell Line , Dystrophin/genetics , Humans , Induced Pluripotent Stem Cells/pathology , Oxidative Stress , Reactive Oxygen Species/metabolism
6.
Nat Cell Biol ; 20(4): 413-421, 2018 04.
Article in English | MEDLINE | ID: mdl-29531308

ABSTRACT

Ageing of haematopoietic stem cells (HSCs) contributes to deficits in the aged haematopoietic system. HSC decline is driven in part by DNA damage accumulation; yet, how ageing impacts the acute DNA damage response (DDR) of HSCs is poorly understood. We show that old HSCs exhibit diminished ATM activity and attenuated DDR, leading to elevated clonal survival in response to a range of genotoxins that was underwritten by diminished apoptotic priming. Distinct HSC subsets exhibited ageing-dependent and subtype-dependent differences in apoptotic priming and survival in response to DNA damage. The defective DDR of old HSCs was non-cell autonomous, as ATM signalling and clonal survival in response to DNA damage could be restored to levels observed in young HSCs post-transplantated into young recipients. These data indicate that defective DDR and diminished apoptotic priming provide a selective advantage to old HSCs that may contribute to mutation accrual and disease predisposition.


Subject(s)
Apoptosis , Cellular Senescence , DNA Damage , DNA Repair , Hematopoietic Stem Cells/enzymology , Age Factors , Animals , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cells, Cultured , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/pathology , Male , Mice, Inbred C57BL , Signal Transduction , Time Factors
7.
Cell Stem Cell ; 22(3): 281-282, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29499142

ABSTRACT

The rapid proliferation and unlimited self-renewal of embryonic stem cells depends upon a permissive chromatin landscape that enables hypertranscription. In this issue of Cell Stem Cell, Bulut-Karslioglu et al. report that euchromatin and transcriptional output are enhanced by protein synthesis in embryonic stem cells (Bulut-Karslioglu et al., 2018).


Subject(s)
Chromatin , Embryonic Stem Cells , Protein Biosynthesis
8.
Stem Cells Dev ; 23(20): 2443-54, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-24836366

ABSTRACT

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.


Subject(s)
Genetic Loci , Hypoxanthine Phosphoribosyltransferase/genetics , Induced Pluripotent Stem Cells/metabolism , Mutation Rate , Cell Differentiation/radiation effects , Cell Line , Gamma Rays , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Induced Pluripotent Stem Cells/cytology
9.
Stem Cells ; 31(4): 693-702, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23315699

ABSTRACT

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.


Subject(s)
DNA Repair/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Embryonic Stem Cells/enzymology , Embryonic Stem Cells/metabolism , Cell Line , DNA Breaks, Double-Stranded , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Humans , Immunohistochemistry , Karyotyping
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