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Proceedings of the National Academy of... Sep 2023Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during...
Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil -glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated -glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of -glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil -glycoproteome featuring intriguing organelle-specific -glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.
Topics: Glycosylation; Neutrophils; Proteome; Cognition; Cytoplasmic Granules
PubMed: 37639587
DOI: 10.1073/pnas.2303867120 -
The Journal of Allergy and Clinical... Jul 2023Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block...
BACKGROUND
Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases.
OBJECTIVE
We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis.
METHODS
Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34 and HL-60 cells.
RESULTS
We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34 cells rescued the promyelocyte differentiation arrest.
CONCLUSION
Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.
Topics: Humans; Cell Cycle Proteins; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae Proteins; Mutation; Phosphorylation
PubMed: 36841265
DOI: 10.1016/j.jaci.2023.02.016 -
Journal of Molecular Biology Aug 2023The cellular defense against viruses involves the assembly of oligomers, granules and membraneless organelles (MLOs) that govern the activation of several arms of the... (Review)
Review
The cellular defense against viruses involves the assembly of oligomers, granules and membraneless organelles (MLOs) that govern the activation of several arms of the innate immune response. Upon interaction with specific pathogen-derived ligands, a number of pattern recognition receptors (PRRs) undergo phase-separation thus triggering downstream signaling pathways. Among other relevant condensates, inflammasomes, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) specks, cyclic GMP-AMP synthase (cGAS) foci, protein kinase R (PKR) clusters, ribonuclease L-induced bodies (RLBs), stress granules (SGs), processing bodies (PBs) and promyelocytic leukemia protein nuclear bodies (PML NBs) play different roles in the immune response. In turn, viruses have evolved diverse strategies to evade the host defense. Viral DNA or RNA, as well as viral proteases or proteins carrying intrinsically disordered regions may interfere with condensate formation and function in multiple ways. In this review we discuss current and hypothetical mechanisms of viral escape that involve the disassembly, repurposing, or inactivation of membraneless condensates that govern innate immunity. We summarize emerging interconnections between these diverse condensates that ultimately determine the cellular outcome.
Topics: Biomolecular Condensates; Immunity, Innate; Signal Transduction; Viruses; Immune Evasion
PubMed: 36702393
DOI: 10.1016/j.jmb.2023.167976 -
Leukemia Sep 2023The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30)...
The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30) isoform. Mutations within the CEBPA gene selectively deleting p42 are frequent in human acute myeloid leukemia. Here we investigated the individual genomics and transcriptomics of p42 and p30. Both proteins bound to identical sites across the genome. For most targets, they induced a highly similar transcriptional response with the exception of a few isoform specific genes. Amongst those we identified early growth response 1 (Egr1) and tribbles1 (Trib1) as key targets selectively induced by p42 that are also underrepresented in CEBPA-mutated AML. Egr1 executed a program of myeloid differentiation and growth arrest. Oppositely, Trib1 established a negative feedback loop through activation of Erk1/2 kinase thus placing differentiation under control of signaling. Unexpectedly, differentiation elicited either by removal of an oncogenic input or by G-CSF did not peruse C/ebpα as mediator but rather directly affected the cell cycle core by upregulation of p21/p27 inhibitors. This points to functions downstream of C/ebpα as intersection point where transforming and differentiation stimuli converge and this finding offers a new perspective for therapeutic intervention.
Topics: Humans; Granulocyte Precursor Cells; Leukemia, Myeloid, Acute; Cell Differentiation; Protein Isoforms; Mutation; CCAAT-Enhancer-Binding Protein-alpha
PubMed: 37532789
DOI: 10.1038/s41375-023-01989-8 -
Cancers Jul 2023Acute promyelocytic leukemia (APL) has a well-established mechanism and a long-term prognosis that exceeds that of any other acute leukemia. These improving outcomes are... (Review)
Review
Acute promyelocytic leukemia (APL) has a well-established mechanism and a long-term prognosis that exceeds that of any other acute leukemia. These improving outcomes are due, in part, to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), two targeted and highly active agents in this disease. However, there remains a considerable morbidity and mortality risk in APL secondary to clinically significant hemorrhagic and/or thrombotic events. Prevention and treatment of these coagulopathic complications remain significant impediments to further progress in optimizing outcomes for patients with APL. Moreover, the relative rarity of APL hinders adequately powered randomized controlled trials for evaluating APL coagulopathy management strategies. This review draws from peer-reviewed works falling between initial descriptions of APL in 1957 and work published prior to January 2023 and provides an updated overview of the pathophysiology of hemorrhagic and thrombotic complications in APL, outlines risk stratification parameters, and compiles current clinical best practices. An improved understanding of the pathophysiologic mechanisms driving hemorrhage and thrombosis along with the completion of well-designed trials of management strategies will assist clinicians in developing interventions that mitigate these devastating complications in an otherwise largely curable disease.
PubMed: 37444587
DOI: 10.3390/cancers15133477 -
International Journal of Hematology May 2024Acute promyelocytic leukemia (APL) is associated with a high incidence of early death, which occurs within 30 days of diagnosis. The major cause of early death in APL... (Review)
Review
Acute promyelocytic leukemia (APL) is associated with a high incidence of early death, which occurs within 30 days of diagnosis. The major cause of early death in APL is severe bleeding, particularly intracranial bleeding. Although APL is known to be associated with activation of coagulation, hyperfibrinolysis, and thrombocytopenia, the precise mechanisms that cause bleeding have not yet been elucidated. I propose that a combination of four pathways may contribute to bleeding in APL: (1) tissue factor, (2) the urokinase plasminogen activator/urokinase plasminogen activator receptor, (3) the annexin A2/S100A100/tissue plasminogen activator, and (4) the podoplanin/C-type lectin-like receptor 2. A better understanding of these pathways will identify new biomarkers to determine which APL patients are at high risk of bleeding and allow the development of new treatments for APL-associated bleeding.
Topics: Humans; Leukemia, Promyelocytic, Acute; Hemostasis; Annexin A2; Hemorrhage; Thromboplastin; Membrane Glycoproteins; Tissue Plasminogen Activator; Receptors, Urokinase Plasminogen Activator; S100 Proteins
PubMed: 38341391
DOI: 10.1007/s12185-024-03708-0 -
International Journal of Molecular... Nov 2023PML bodies are subnuclear protein complexes that play a crucial role in various physiological and pathological cellular processes. One of the general structural proteins... (Review)
Review
PML bodies are subnuclear protein complexes that play a crucial role in various physiological and pathological cellular processes. One of the general structural proteins of PML bodies is a member of the tripartite motif (TRIM) family-promyelocytic leukemia protein (PML). It is known that PML interacts with over a hundred partners, and the protein itself is represented by several major isoforms, differing in their variable and disordered C-terminal end due to alternative splicing. Despite nearly 30 years of research, the mechanisms underlying PML body formation and the role of PML proteins in this process remain largely unclear. In this review, we examine the literature and highlight recent progress in this field, with a particular focus on understanding the role of individual domains of the PML protein, its post-translational modifications, and polyvalent nonspecific interactions in the formation of PML bodies. Additionally, based on the available literature, we propose a new hypothetical model of PML body formation.
Topics: Nuclear Proteins; Promyelocytic Leukemia Nuclear Bodies; Tumor Suppressor Proteins; Promyelocytic Leukemia Protein; Transcription Factors; Tripartite Motif Proteins
PubMed: 38069029
DOI: 10.3390/ijms242316702 -
Neuron May 2024Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to...
Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to study the cell-type-specific effects of glucocorticoids on neurogenesis. We show that glucocorticoids increase a specific type of basal progenitors (co-expressing PAX6 and EOMES) that has been shown to contribute to cortical expansion in gyrified species. This effect is mediated via the transcription factor ZBTB16 and leads to increased production of neurons. A phenome-wide Mendelian randomization analysis of an enhancer variant that moderates glucocorticoid-induced ZBTB16 levels reveals causal relationships with higher educational attainment and altered brain structure. The relationship with postnatal cognition is also supported by data from a prospective pregnancy cohort study. This work provides a cellular and molecular pathway for the effects of glucocorticoids on human neurogenesis that relates to lasting postnatal phenotypes.
Topics: Neurogenesis; Humans; Animals; Mice; Glucocorticoids; Cerebral Cortex; Female; Promyelocytic Leukemia Zinc Finger Protein; Pregnancy; Neurons; Organoids; Gene Expression Regulation, Developmental; Neural Stem Cells; Male
PubMed: 38442714
DOI: 10.1016/j.neuron.2024.02.005 -
Cancers Jul 2023Since the introduction of all-trans retinoic acid (ATRA), acute promyelocytic leukemia (APL) has become a highly curable malignancy, especially in combination with... (Review)
Review
Since the introduction of all-trans retinoic acid (ATRA), acute promyelocytic leukemia (APL) has become a highly curable malignancy, especially in combination with arsenic trioxide (ATO). ATRA's success has deepened our understanding of the role of the RARα pathway in normal hematopoiesis and leukemogenesis, and it has influenced a generation of cancer drug development. Retinoids have also demonstrated some efficacy in a handful of other disease entities, including as a maintenance therapy for neuroblastoma and in the treatment of cutaneous T-cell lymphomas; nevertheless, the promise of retinoids as a differentiating therapy in acute myeloid leukemia (AML) more broadly, and as a cancer preventative, have largely gone unfulfilled. Recent research into the mechanisms of ATRA resistance and the biomarkers of RARα pathway dysregulation in AML have reinvigorated efforts to successfully deploy retinoid therapy in a broader subset of myeloid malignancies. Recent studies have demonstrated that the bone marrow environment is highly protected from exogenous ATRA via local homeostasis controlled by stromal cells expressing CYP26, a key enzyme responsible for ATRA inactivation. Synthetic CYP26-resistant retinoids such as tamibarotene bypass this stromal protection and have shown superior anti-leukemic effects. Furthermore, recent super-enhancer (SE) analysis has identified a novel AML subgroup characterized by high expression of RARα through strong SE levels in the gene locus and increased sensitivity to tamibarotene. Combined with a hypomethylating agent, synthetic retinoids have shown synergistic anti-leukemic effects in non-APL AML preclinical models and are now being studied in phase II and III clinical trials.
PubMed: 37509198
DOI: 10.3390/cancers15143535 -
Leukemia Aug 2023We characterize the metabolic background in distinct Acute Myeloid Leukemias (AMLs), by comparing the metabolism of primary AML blasts isolated at diagnosis with that of...
We characterize the metabolic background in distinct Acute Myeloid Leukemias (AMLs), by comparing the metabolism of primary AML blasts isolated at diagnosis with that of normal hematopoietic maturing progenitors, using the Seahorse XF Agilent. Leukemic cells feature lower spare respiratory (SRC) and glycolytic capacities as compared to hematopoietic precursors (i.e. day 7, promyelocytes). According with Proton Leak (PL) values, AML blasts can be grouped in two well defined populations. The AML group with blasts presenting high PL or high basal OXPHOS plus high SRC levels had shorter overall survival time and significantly overexpressed myeloid cell leukemia 1 (MCL1) protein. We demonstrate that MCL1 directly binds to Hexokinase 2 (HK2) on the outer mitochondrial membrane (OMM). Overall, these results suggest that high PL and high SRC plus high basal OXPHOS levels at disease onset, arguably with the concourse of MCL1/HK2 action, are significantly linked with shorter overall survival time in AML. Our data describe a new function for MCL1 protein in AMLs' cells: by forming a complex with HK2, MCL1 co-localizes to VDAC on the OMM, thus inducing glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy.
Topics: Humans; Hexokinase; Leukemia, Myeloid, Acute; Myeloid Cell Leukemia Sequence 1 Protein
PubMed: 37349598
DOI: 10.1038/s41375-023-01946-5