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1.
Res Sq ; 2024 May 30.
Article in English | MEDLINE | ID: mdl-38853828

ABSTRACT

Aging is a prominent risk factor for Alzheimer's disease (AD), but the cellular mechanisms underlying neuronal phenotypes remain elusive. Both accumulation of amyloid plaques and neurofibrillary tangles in the brain1 and age-linked organelle deficits2-7 are proposed as causes of AD phenotypes but the relationship between these events is unclear. Here, we address this question using a transdifferentiated neuron (tNeuron) model directly from human dermal fibroblasts. Patient-derived tNeurons retain aging hallmarks and exhibit AD-linked deficits. Quantitative tNeuron proteomic analyses identify aging and AD-linked deficits in proteostasis and organelle homeostasis, particularly affecting endosome-lysosomal components. The proteostasis and lysosomal homeostasis deficits in aged tNeurons are exacerbated in sporadic and familial AD tNeurons, promoting constitutive lysosomal damage and defects in ESCRT-mediated repair. We find deficits in neuronal lysosomal homeostasis lead to inflammatory cytokine secretion, cell death and spontaneous development of Aß and phospho-Tau deposits. These proteotoxic inclusions co-localize with lysosomes and damage markers and resemble inclusions in brain tissue from AD patients and APP-transgenic mice. Supporting the centrality of lysosomal deficits driving AD phenotypes, lysosome-function enhancing compounds reduce AD-associated cytokine secretion and Aß deposits. We conclude that proteostasis and organelle deficits are upstream initiating factors leading to neuronal aging and AD phenotypes.

2.
Nature ; 630(8015): 198-205, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38720074

ABSTRACT

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.


Subject(s)
Class Ib Phosphatidylinositol 3-Kinase , Leukemia , Signal Transduction , p21-Activated Kinases , Animals , Humans , Mice , Cell Line , Class Ib Phosphatidylinositol 3-Kinase/genetics , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Cytarabine/pharmacology , Cytarabine/therapeutic use , Leukemia/drug therapy , Leukemia/enzymology , Leukemia/genetics , Leukemia/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Oxidative Phosphorylation/drug effects , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/metabolism , Phosphorylation , Xenograft Model Antitumor Assays
3.
Genet Med ; 26(6): 101120, 2024 06.
Article in English | MEDLINE | ID: mdl-38469793

ABSTRACT

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.


Subject(s)
Neurodevelopmental Disorders , Humans , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Female , Male , Alleles , Autophagy/genetics , Ubiquitin Thiolesterase/genetics , Infant, Newborn , Proteasome Endopeptidase Complex/genetics , Pedigree , Homozygote , Genetic Predisposition to Disease , Mutation/genetics
4.
bioRxiv ; 2023 Mar 27.
Article in English | MEDLINE | ID: mdl-37034684

ABSTRACT

The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer's disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-ß inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

5.
bioRxiv ; 2023 Jan 20.
Article in English | MEDLINE | ID: mdl-36712069

ABSTRACT

Haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), a devastating neurodegenerative disease with no effective treatment. PGRN is required for efficient proteostasis, as loss of neuronal PGRN results in dysfunctional lysosomes and impaired clearance and cytoplasmic aggregation of TDP-43, a protein involved in neurodegeneration in FTD. These and other events lead to neurodegeneration and neuroinflammation. However, the detailed mechanisms leading to protein dyshomeostasis in PGRN-deficient cells remain unclear. We report here the development of human cell models of FTD with PGRN-deficiency to explore the molecular mechanisms underlying proteostasis breakdown and TDP-43 aggregation in FTD. Neurons differentiated from FTD patient induced pluripotent stem cells (iPSCs) have reduced PGRN levels, and the neurons recapitulate key disease features, including impaired lysosomal function, defective TDP-43 turnover and accumulation, neurodegeneration, and death. Proteomic analysis revealed altered levels of proteins linked to the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) in FTD patient neurons, providing new mechanistic insights into the link between PGRN-deficiency and disease pathobiology.

6.
J Clin Invest ; 133(6)2023 03 15.
Article in English | MEDLINE | ID: mdl-36719747

ABSTRACT

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.


Subject(s)
Myeloproliferative Disorders , Neoplasms , Mice , Animals , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Cell Proliferation , Janus Kinase 2/metabolism
7.
bioRxiv ; 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-38328043

ABSTRACT

Phosphoinositide 3-kinase gamma (PI3Kγ) is implicated as a target to repolarize tumor-associated macrophages and promote anti-tumor immune responses in solid cancers. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid, and dendritic lineages. This dependency is characterized by innate inflammatory signaling and activation of phosphoinositide 3-kinase regulatory subunit 5 ( PIK3R5 ), which encodes a regulatory subunit of PI3Kγ and stabilizes the active enzymatic complex. Mechanistically, we identify p21 (RAC1) activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency independently of Akt kinase. PI3Kγ inhibition dephosphorylates PAK1, activates a transcriptional network of NFκB-related tumor suppressor genes, and impairs mitochondrial oxidative phosphorylation. We find that treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukemias with activated PIK3R5 , either at baseline or by exogenous inflammatory stimulation. Notably, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukemia xenografts with low baseline PIK3R5 expression, as residual leukemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Taken together, our study reveals a targetable dependency on PI3Kγ/PAK1 signaling that is amenable to near-term evaluation in patients with acute leukemia.

8.
Nat Metab ; 4(12): 1812-1829, 2022 12.
Article in English | MEDLINE | ID: mdl-36536133

ABSTRACT

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.


Subject(s)
Alternative Splicing , RNA-Binding Proteins , Mice , Animals , Alternative Splicing/genetics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/genetics , Liver/metabolism , Homeostasis , Cholesterol/metabolism , Scavenger Receptors, Class B/genetics , Scavenger Receptors, Class B/metabolism
9.
Nat Struct Mol Biol ; 28(5): 418-425, 2021 05.
Article in English | MEDLINE | ID: mdl-33846632

ABSTRACT

The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7ß7ß7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial ß-ring (ß2-4), Ump1 and Pba1/2. The second structure contains two additional subunits (ß5-6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and ß2/ß5 propeptides, with important implications for their functions. Unexpectedly, Pba1's N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the ß5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.


Subject(s)
Molecular Chaperones , Proteasome Endopeptidase Complex , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Catalytic Domain , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Conformation , Protein Subunits , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
10.
Hum Mol Genet ; 30(13): 1230-1246, 2021 06 17.
Article in English | MEDLINE | ID: mdl-33891006

ABSTRACT

UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44-targeting defects were rescued by reexpression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Amyotrophic Lateral Sclerosis/genetics , Autophagy-Related Proteins/genetics , Frontotemporal Dementia/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Mutation , Animals , Cell Line , Disease Models, Animal , HeLa Cells , Humans , Immunoblotting , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Electron, Transmission , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Proteins/metabolism , Oxygen Consumption/genetics , Proteomics/methods
11.
J Nutr ; 151(5): 1073-1083, 2021 05 11.
Article in English | MEDLINE | ID: mdl-33693820

ABSTRACT

BACKGROUND: Maternal iron deficiency (ID) is associated with poor pregnancy and fetal outcomes. The effect is thought to be mediated by the placenta but there is no comprehensive assessment of placental responses to maternal ID. Additionally, whether the influence of maternal ID on the placenta differs by fetal sex is unknown. OBJECTIVES: To identify gene and protein signatures of ID mouse placentas at mid-gestation. A secondary objective was to profile the expression of iron genes in mouse placentas across gestation. METHODS: We used a real-time PCR-based array to determine the mRNA expression of all known iron genes in mouse placentas at embryonic day (E) 12.5, E14.5, E16.5, and E19.5 (n = 3 placentas/time point). To determine the effect of maternal ID, we performed RNA sequencing and proteomics in male and female placentas from ID and iron-adequate mice at E12.5 (n = 8 dams/diet). RESULTS: In female placentas, 6 genes, including transferrin receptor (Tfrc) and solute carrier family 11 member 2, were significantly changed by maternal ID. An additional 154 genes were altered in male ID placentas. A proteomic analysis quantified 7662 proteins in the placenta. Proteins translated from iron-responsive element (IRE)-containing mRNA were altered in abundance; ferritin and ferroportin 1 decreased, while TFRC increased in ID placentas. Less than 4% of the significantly altered genes in ID placentas occurred both at the transcriptional and translational levels. CONCLUSIONS: Our data demonstrate that the impact of maternal ID on placental gene expression in mice is limited in scope and magnitude at mid-gestation. We provide strong evidence for IRE-based transcriptional and translational coordination of iron gene expression in the mouse placenta. Finally, we discover sexually dimorphic effects of maternal ID on placental gene expression, with more genes and pathways altered in male compared with female mouse placentas.


Subject(s)
Anemia, Iron-Deficiency/metabolism , Placenta/metabolism , Pregnancy Complications/metabolism , Proteome/metabolism , Transcriptome/physiology , Animals , Female , Gene Expression Regulation , Iron/metabolism , Iron/pharmacology , Mice , Nonheme Iron Proteins/genetics , Nonheme Iron Proteins/metabolism , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolism
12.
J Biol Chem ; 296: 100153, 2021.
Article in English | MEDLINE | ID: mdl-33277362

ABSTRACT

Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Amyotrophic Lateral Sclerosis/genetics , Autophagy-Related Proteins/genetics , Frontotemporal Dementia/genetics , Proteasome Endopeptidase Complex/metabolism , Proteomics/methods , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Proteins/deficiency , Autophagy-Related Proteins/metabolism , Cell Line , Cycloheximide/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Gene Expression Regulation , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Protein Stability/drug effects , Proteolysis/drug effects , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Serotonin/metabolism , Signal Transduction , Trans-Activators/genetics , Trans-Activators/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
13.
Blood ; 137(3): 398-409, 2021 01 21.
Article in English | MEDLINE | ID: mdl-33036023

ABSTRACT

The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.


Subject(s)
Erythroblasts/metabolism , Erythrocyte Membrane/metabolism , Organelles/metabolism , Proteome/metabolism , Animals , Autophagosomes/metabolism , Base Sequence , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomes/metabolism , Erythroblasts/ultrastructure , Erythrocyte Membrane/ultrastructure , Erythropoiesis , Lysosomes/metabolism , Membrane Fusion , Mice, Inbred C57BL , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/metabolism , NADPH Dehydrogenase/deficiency , NADPH Dehydrogenase/metabolism , Organelles/ultrastructure , Reticulocytes/metabolism , Reticulocytes/ultrastructure
14.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Article in English | MEDLINE | ID: mdl-32513711

ABSTRACT

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Amyotrophic Lateral Sclerosis/genetics , Autophagosomes/physiology , Autophagy-Related Proteins/metabolism , Autophagy/genetics , Dementia/genetics , Adaptor Proteins, Signal Transducing/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Autophagy-Related Proteins/genetics , Biomarkers/metabolism , Cell Line , Dementia/metabolism , Dementia/pathology , Genetic Predisposition to Disease , Humans , Hydrogen-Ion Concentration , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Mice , Mice, Transgenic , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mutation , Protein Binding , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Up-Regulation , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism
15.
Nat Commun ; 11(1): 1406, 2020 03 16.
Article in English | MEDLINE | ID: mdl-32179749

ABSTRACT

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.


Subject(s)
Chromatin/metabolism , Hematopoietic Stem Cells/metabolism , Leukemia, Myeloid, Acute/metabolism , Acetylation , Animals , Cell Differentiation , Cell Survival , Female , HMGN1 Protein/genetics , HMGN1 Protein/metabolism , Hematopoietic Stem Cells/cytology , Histones/genetics , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism
16.
Article in English | MEDLINE | ID: mdl-30833452

ABSTRACT

The proteasome, the most complex protease known, degrades proteins that have been conjugated to ubiquitin. It faces the unique challenge of acting enzymatically on hundreds and perhaps thousands of structurally diverse substrates, mechanically unfolding them from their native state and translocating them vectorially from one specialized compartment of the enzyme to another. Moreover, substrates are modified by ubiquitin in myriad configurations of chains. The many unusual design features of the proteasome may have evolved in part to endow this enzyme with a robust ability to process substrates regardless of their identity. The proteasome plays a major role in preserving protein homeostasis in the cell, which requires adaptation to a wide variety of stress conditions. Modulation of proteasome function is achieved through a large network of proteins that interact with it dynamically, modify it enzymatically, or fine-tune its levels. The resulting adaptability of the proteasome, which is unique among proteases, enables cells to control the output of the ubiquitin-proteasome pathway on a global scale.


Subject(s)
Gene Expression Regulation , Proteasome Endopeptidase Complex/chemistry , Protein Engineering/methods , Ubiquitin/chemistry , Adenosine Triphosphate/chemistry , Animals , Caenorhabditis elegans , Cryoelectron Microscopy , Cytoplasm/metabolism , DNA-Binding Proteins/chemistry , Homeostasis , Humans , Models, Molecular , Molecular Conformation , Nuclear Respiratory Factor 1/chemistry , Protein Denaturation , Protein Folding , Protein Processing, Post-Translational , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Transcription Factors/chemistry , Ubiquitin Thiolesterase/chemistry
17.
Sci Signal ; 12(609)2019 11 26.
Article in English | MEDLINE | ID: mdl-31772124

ABSTRACT

The yeast stress-activated protein kinase Hog1 is best known for its role in mediating the response to osmotic stress, but it is also activated by various mechanistically distinct environmental stressors, including heat shock, endoplasmic reticulum stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 through a kinase cascade. Here, we identified a mode of Hog1 function whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop. These residues were essential for Hog1-mediated protection against arsenic, were dispensable for the response to osmotic stress, and promoted the nuclear localization of Hog1 upon exposure of cells to arsenic. Hog1 promoted arsenic detoxification by stimulating phosphorylation of the transcription factor Yap8, promoting Yap8 nuclear localization, and stimulating the transcription of the only known Yap8 targets, ARR2 and ARR3, both of which encode proteins that promote arsenic efflux. The related human kinases ERK1 and ERK2 also bound to arsenic in vitro, suggesting that this may be a conserved feature of some members of the mitogen-activated protein kinase (MAPK) family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses.


Subject(s)
Arsenic/pharmacology , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Arsenate Reductases/genetics , Arsenate Reductases/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
18.
Hum Mol Genet ; 28(5): 764-777, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30388222

ABSTRACT

Primary cilia are hair-like organelles that play crucial roles in vertebrate development, organogenesis and when dysfunctional result in pleiotropic human genetic disorders called ciliopathies, characterized by overlapping phenotypes, such as renal and hepatic cysts, skeletal defects, retinal degeneration and central nervous system malformations. Primary cilia act as communication hubs to transfer extracellular signals into intracellular responses and are essential for Hedgehog (Hh) signal transduction in mammals. Despite the renewed interest in this ancient organelle of growing biomedical importance, the molecular mechanisms that trigger cilia formation, extension and ciliary signal transduction are still not fully understood. Here we provide, for the first time, evidence that the deubiquitinase ubiquitin-specific protease-14 (Usp14), a major regulator of the ubiquitin proteasome system (UPS), controls ciliogenesis, cilia elongation and Hh signal transduction. Moreover, we show that pharmacological inhibition of Usp14 positively affects Hh signal transduction in a model of autosomal dominant polycystic kidney disease. These findings provide new insight into the spectrum of action of UPS in cilia biology and may provide novel opportunities for therapeutic intervention in human conditions associated with ciliary dysfunction.


Subject(s)
Cilia/metabolism , Hedgehog Proteins/metabolism , Organogenesis/genetics , Signal Transduction , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Animals , Biomarkers , Cell Line , Deubiquitinating Enzymes/genetics , Deubiquitinating Enzymes/metabolism , Fibroblasts , Fluorescent Antibody Technique , Gene Expression Regulation , Mice , Mutation , Protein Transport , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism
19.
Blood ; 132(22): 2375-2388, 2018 11 29.
Article in English | MEDLINE | ID: mdl-30181176

ABSTRACT

Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Ribosomal Proteins/genetics , Ribosomes/genetics , Cell Line, Tumor , Cohort Studies , HEK293 Cells , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mutation Rate , Point Mutation , Protein Biosynthesis , Protein Domains , Ribosomal Proteins/chemistry , Ribosomes/pathology
20.
J Biol Chem ; 293(6): 2183-2194, 2018 02 09.
Article in English | MEDLINE | ID: mdl-29273634

ABSTRACT

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.


Subject(s)
Blastocyst , DEAD-box RNA Helicases/chemistry , Deubiquitinating Enzymes/physiology , RNA Helicases/chemistry , Ubiquitin Thiolesterase/physiology , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Deubiquitinating Enzymes/genetics , Embryo Loss , Humans , Mice , Mice, Knockout , Protein Biosynthesis , Protein Stability , RNA, Ribosomal , Ubiquitin Thiolesterase/genetics
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