LMAN1 serves as a cargo receptor for thrombopoietin.

Thrombopoietin (TPO) is a plasma glycoprotein that binds its receptor on megakaryocytes (MKs) and MK progenitors, resulting in enhanced platelet production. The mechanism by which TPO is secreted from hepatocytes remains poorly understood. Lectin mannose-binding 1 (LMAN1) and multiple coagulation factor deficiency 2 (MCFD2) form a complex at the endoplasmic reticulum membrane, recruiting cargo proteins into COPII vesicles for secretion.

TR4 and BCL11A Repress γ-globin Transcription via Independent Mechanisms.

Nuclear receptor TR4 was previously shown to bind to the -117 position of the -globin gene promoters in vitro, which overlaps the more recently described BCL11A binding site. The role of TR4 in human -globin gene repression has not been extensively characterized in vivo, while any relationship between TR4 and BCL11A regulation through the -globin promoters is unclear at present. We show here that TR4 and BCL11A competitively bind in vitro to distinct, overlapping sequences, including positions overlapping -117 of the -globin promoter.

Functional overlap between the mammalian and paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two paralogs, and .

Functional overlap between the mammalian and paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two paralogs, and .

Identification of LMAN1- and SURF4-Dependent Secretory Cargoes.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the or gene.

Murine erythroid differentiation kinetics in vivo under normal and anemic stress conditions.

Our current understanding of the kinetics and dynamics of erythroid differentiation is based almost entirely on the ex vivo expansion of cultured hematopoietic progenitor cells. In this study, we used an erythroid-specific, inducible transgenic mouse line to investigate for the first time, the in vivo erythroid differentiation kinetics under steady-state conditions. We demonstrated that bipotent premegakaroycyte/erythroid (PreMegE) progenitor cells differentiate into erythroid-committed proerythroblast/basophilic erythroblasts (ProBasoE) after 6.

Hepatic inactivation of murine results in marked reduction in plasma cholesterol.

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018).

The congenital dyserythropoieitic anemias: genetics and pathophysiology.

Purpose Of Review: The congenital dyserythropoietic anemias (CDA) are hereditary disorders characterized by ineffective erythropoiesis. This review evaluates newly developed CDA disease models, the latest advances in understanding the pathogenesis of the CDAs, and recently identified CDA genes.

Recent Findings: Mice exhibiting features of CDAI were recently generated, demonstrating that Codanin-1 (encoded by Cdan1) is essential for primitive erythropoiesis.

Identification of novel γ-globin inducers among all potential erythroid druggable targets.

Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited β-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals.

A common human missense mutation of vesicle coat protein SEC23B leads to growth restriction and chronic pancreatitis in mice.

SEC23B is one of two vertebrate paralogs of SEC23, a key component of the coat protein complex II vesicles. Complete deficiency of SEC23B in mice leads to perinatal death caused by massive degeneration of professional secretory tissues. However, functions of SEC23B in postnatal mice and outside professional secretory tissues are unclear.

SEC23A rescues SEC23B-deficient congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDAII) results from loss-of-function mutations in . In contrast to humans, SEC23B-deficient mice deletion do not exhibit CDAII but die perinatally with pancreatic degeneration. Here, we demonstrate that expression of the full SEC23A protein (the SEC23B paralog) from the endogenous regulatory elements of completely rescues the SEC23B-deficient mouse phenotype.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.

Epidemiologic and Genetic Associations of Erythropoietin With Blood Pressure, Hypertension, and Coronary Artery Disease.

[Figure: see text].

A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia.

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins are found in most individuals with Diamond-Blackfan anemia (DBA), whereas somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human patients with DBA.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene.

TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis.

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining.

ER-to-Golgi transport and SEC23-dependent COPII vesicles regulate T cell alloimmunity.

T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway.

Altered phenotype in LMAN1-deficient mice with low levels of residual LMAN1 expression.

Combined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder caused by loss-of-function mutations in either LMAN1 or MCFD2. The latter genes encode 2 components of a mammalian cargo receptor that facilitates secretion of coagulation factor V (FV) and factor VIII (FVIII) from the endoplasmic reticulum (ER) to the Golgi via coat protein complex II vesicles. F5F8D patients exhibit FV and FVIII levels that are ∼10% to 15% of normal.

The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.

Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion.

Role of caplacizumab in the treatment of acquired thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura is a rare blood disorder with a high early mortality rate, if untreated. Standard of care plasma exchange and glucocorticoids have dramatically improved survival. However, additional advancements are necessary to further decrease mortality.

Murine Surf4 is essential for early embryonic development.

Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4.

SNARE protein SEC22B regulates early embryonic development.

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo.

Genome-wide linkage analysis and whole-exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia.

Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect.

Impaired human hematopoiesis due to a cryptic intronic splicing mutation.

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging.

A diagnosis of discernment: Identifying a novel ATRX mutation in myelodysplastic syndrome with acquired α-thalassemia.

Myelodysplastic syndromes (MDS) are a heterogeneous category of myeloid neoplasms that represent the most common class of acquired bone marrow failure syndromes in adults. MDS is typically associated with a hypoproliferative macrocytic anemia, but atypical findings on initial diagnostic evaluations can raise concern for a distinct pathophysiological process and lead to the investigation of alternative etiologies. Here, we report a case of MDS with a concomitant hypoproliferative microcytic and hypochromic anemia that led to the identification of acquired hemoglobin H due to a novel somatic ATRX mutation.