Anchoring of FLT3 in the endoplasmic reticulum alters signaling quality

Abstract The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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Antioxidant Therapy in Graves’ Orbitopathy

Frontiers in Endocrinology ◽

10.3389/fendo.2020.608733 ◽

2020 ◽

Vol 11 ◽

Author(s):

Giulia Lanzolla ◽

Claudio Marcocci ◽

Michele Marinò

Keyword(s):

Ros Generation ◽

Related Condition ◽

Orbital Radiotherapy ◽

Antioxidant Agents ◽

Graves Orbitopathy ◽

High Doses

The balance of the cell redox state is a key point for the maintenance of cellular homeostasis. Increased reactive oxygen species (ROS) generation leads to oxidative damage of tissues, which is involved in the development of several diseases, including autoimmune diseases. Graves’ Orbitopathy (GO) is a disfiguring autoimmune-related condition associated with Graves’ Disease (GD). Patients with active, moderate-to-severe GO, are generally treated with high doses intravenous glucocorticoids (ivGCs) and/or orbital radiotherapy. On the contrary, up to recently, local ointments were the treatment most frequently offered to patients with mild GO, because the risks related to ivGCs does not justify the relatively poor benefits expected in mild GO. However, a medical treatment for these patients is heavily wanted, considering that GO can progress into more severe forms and also patients with mild GO complain with an impairment in their quality of life. Thus, based on the role of oxidative stress in the pathogenesis of GO, a therapy with antioxidant agents has been proposed and a number of studies have been performed, both in vitro and in vivo, which is reviewed here.

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Targeting Wnt/β-Catenin Pathway for Developing Therapies for Hair Loss

International Journal of Molecular Sciences ◽

10.3390/ijms21144915 ◽

2020 ◽

Vol 21 (14) ◽

pp. 4915 ◽

Cited By ~ 1

Author(s):

Bu Young Choi

Keyword(s):

Hair Loss ◽

Hair Growth ◽

In Vivo Studies ◽

Intracellular Targets ◽

Remarkable Progress ◽

Made In

Persistent hair loss is a major cause of psychological distress and compromised quality of life in millions of people worldwide. Remarkable progress has been made in understanding the molecular basis of hair loss and identifying valid intracellular targets for designing effective therapies for hair loss treatment. Whereas a variety of growth factors and signaling pathways have been implicated in hair cycling process, the activation of Wnt/β-catenin signaling plays a central role in hair follicle regeneration. Several plant-derived chemicals have been reported to promote hair growth by activating Wnt/β-catenin signaling in various in vitro and in vivo studies. This mini-review sheds light on the role of Wnt/β-catenin in promoting hair growth and the current progress in designing hair loss therapies by targeting this signaling pathway.

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A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽

10.1182/blood.v120.21.2332.2332 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2332-2332

Author(s):

Vitalyi Senyuk ◽

Yunyuan Zhang ◽

Yang Liu ◽

Ming Ming ◽

Jianjun Chen ◽

...

Keyword(s):

Cell Transformation ◽

Conflicts Of Interest ◽

Critical Role ◽

Ectopic Expression ◽

Myeloid Differentiation ◽

Dna Hypermethylation ◽

Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6923-6934.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6923-6934 ◽

Cited By ~ 64

Author(s):

Mehdi Kabani ◽

Jean-Marie Beckerich ◽

Claude Gaillardin

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Synthetic Lethality ◽

In Vitro Assays ◽

Endoplasmic Reticulum Membrane ◽

Dependent Manner ◽

Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

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Estrogen is required for maintaining the quality of cardiac stem cells

PLoS ONE ◽

10.1371/journal.pone.0245166 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245166

Author(s):

Al Shaimaa Hasan ◽

Lan Luo ◽

Satoko Baba ◽

Tao-Sheng Li

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Potential Role ◽

Telomerase Activity ◽

Estrogen Deficiency ◽

Cardiac Stem Cells

Compared to the age-matched men, the incidence of cardiovascular diseases is lower in premenopausal but higher in postmenopausal women, suggesting the cardio-protective role of estrogen in females. Although cardiac stem cells (CSCs) express estrogen receptors, yet the effects of estrogen on CSCs remain unclear. In this study, we investigated the potential role of estrogen in maintaining the quality of CSCs by in vivo and in vitro experiments. For the in vivo study, estrogen deficiency was induced by ovariectomy in 6-weeks-old C57BL/6 female mice, and then randomly given 17β-estradiol (E2) replacements at a low dose (0.01 mg/60 days) and high dose (0.18 mg/60 days), or vehicle treatment. All mice were killed 2 months after treatments, and heart tissues were collected for ex vivo expansion of CSCs. Compared to age-matched healthy controls, estrogen deficiency slightly decreased the yield of CSCs with significantly lower telomerase activity and more DNA damage. Interestingly, E2 replacements at low and high doses significantly increased the yield of CSCs and reversed the quality impairment of CSCs following estrogen deficiency. For the in vitro study, twice-passaged CSCs from the hearts of adult healthy female mice were cultured with the supplement of 0.01, 0.1, and 1 μM E2 in the medium for 3 days. We found that E2 supplement increased c-kit expression, increased proliferative activity, improved telomerase activity, and reduced DNA damage of CSCs in a dose-dependent manner. Our data suggested the potential role of estrogen in maintaining the quality of CSCs, providing new insight into the cardio-protective effects of estrogen.

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Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

The Journal of Cell Biology ◽

10.1083/jcb.153.5.1061 ◽

2001 ◽

Vol 153 (5) ◽

pp. 1061-1070 ◽

Cited By ~ 240

Author(s):

Shuh-ichi Nishikawa ◽

Sheara W. Fewell ◽

Yoshihito Kato ◽

Jeffrey L. Brodsky ◽

Toshiya Endo

Keyword(s):

Endoplasmic Reticulum ◽

Molecular Chaperone ◽

Molecular Chaperones ◽

Elevated Temperatures ◽

Carboxypeptidase Y ◽

Mutant Form ◽

Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

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Nutraceutical Compounds as Sensitizers for Cancer Treatment in Radiation Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms20215267 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5267 ◽

Cited By ~ 3

Author(s):

Marco Calvaruso ◽

Gaia Pucci ◽

Rosa Musso ◽

Valentina Bravatà ◽

Francesco P. Cammarata ◽

...

Keyword(s):

Cell Damage ◽

Chemical Compounds ◽

Diagnostic Techniques ◽

Response To Treatment ◽

Nutraceutical Compounds ◽

Treatment Plans

The improvement of diagnostic techniques and the efficacy of new therapies in clinical practice have allowed cancer patients to reach a higher chance to be cured together with a better quality of life. However, tumors still represent the second leading cause of death worldwide. On the contrary, chemotherapy and radiotherapy (RT) still lack treatment plans which take into account the biological features of tumors and depend on this for their response to treatment. Tumor cells’ response to RT is strictly-connected to their radiosensitivity, namely, their ability to resist and to overcome cell damage induced by ionizing radiation (IR). For this reason, radiobiological research is focusing on the ability of chemical compounds to radiosensitize cancer cells so to make them more responsive to IR. In recent years, the interests of researchers have been focused on natural compounds that show antitumoral effects with limited collateral issues. Moreover, nutraceuticals are easy to recover and are thus less expensive. On these bases, several scientific projects have aimed to test also their ability to induce tumor radiosensitization both in vitro and in vivo. The goal of this review is to describe what is known about the role of nutraceuticals in radiotherapy, their use and their potential application.

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PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus

Scientific Reports ◽

10.1038/s41598-019-55866-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 10

Author(s):

Mariam Lofty Khaled ◽

Yelena Bykhovskaya ◽

Chunfang Gu ◽

Alice Liu ◽

Michelle D. Drewry ◽

...

Keyword(s):

Gene Trap ◽

Whole Exome ◽

The Us ◽

Related Quality ◽

First Time

AbstractKeratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

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AAV-mediated ERdj5 overexpression protects against P23H rhodopsin toxicity

Human Molecular Genetics ◽

10.1093/hmg/ddaa049 ◽

2020 ◽

Vol 29 (8) ◽

pp. 1310-1318 ◽

Cited By ~ 2

Author(s):

Monica Aguilà ◽

James Bellingham ◽

Dimitra Athanasiou ◽

Dalila Bevilacqua ◽

Yanai Duran ◽

...

Keyword(s):

Quality Control ◽

Outer Nuclear Layer ◽

Photoreceptor Cells ◽

Er Quality Control ◽

Full Field ◽

Er Retention ◽

Function Loss

Abstract Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5−/− and P23H+/−:Erdj5−/− mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

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