Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium.

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

LRP2 contributes to planar cell polarity-dependent coordination of motile cilia function.

Motile cilia are protruding organelles on specialized epithelia that beat in a synchronous fashion to propel extracellular fluids. Coordination and orientation of cilia beating on individual cells and across tissues is a complex process dependent on planar cell polarity (PCP) signaling. Asymmetric sorting of PCP pathway components, essential to establish planar polarity, involves trafficking along the endocytic path, but the underlying regulatory processes remain incompletely understood. Here, we identified the endocytic receptor LRP2 as regulator of PCP component trafficking in ependyma, a multi-ciliated cell type that is involved in facilitating flow of the cerebrospinal fluid in the brain ventricular system. Lack of receptor expression in gene-targeted mice results in a failure to sort PCP core proteins to the anterior or posterior cell side and, consequently, in the inability to coordinate cilia arrangement and to aligned beating (loss of rotational and translational polarity). LRP2 deficiency coincides with a failure to sort NHERF1, a cytoplasmic LRP2 adaptor to the anterior cell side. As NHERF1 is essential to translocate PCP core protein Vangl2 to the plasma membrane, these data suggest a molecular mechanism whereby LRP2 interacts with PCP components through NHERF1 to control their asymmetric sorting along the endocytic path. Taken together, our findings identified the endocytic receptor LRP2 as a novel regulator of endosomal trafficking of PCP proteins, ensuring their asymmetric partition and establishment of translational and rotational planar cell polarity in the ependyma.

LRP2 controls sonic hedgehog-dependent differentiation of cardiac progenitor cells during outflow tract formation.

Conotruncal malformations are a major cause of congenital heart defects in newborn infants. Recently, genetic screens in humans and in mouse models have identified mutations in LRP2, a multi-ligand receptor, as a novel cause of a common arterial trunk, a severe form of outflow tract (OFT) defect. Yet, the underlying mechanism why the morphogen receptor LRP2 is essential for OFT development remained unexplained. Studying LRP2-deficient mouse models, we now show that LRP2 is expressed in the cardiac progenitor niche of the anterior second heart field (SHF) that contributes to the elongation of the OFT during separation into aorta and pulmonary trunk. Loss of LRP2 in mutant mice results in the depletion of a pool of sonic hedgehog-dependent progenitor cells in the anterior SHF due to premature differentiation into cardiomyocytes as they migrate into the OFT myocardium. Depletion of this cardiac progenitor cell pool results in aberrant shortening of the OFT, the likely cause of CAT formation in affected mice. Our findings identified the molecular mechanism whereby LRP2 controls the maintenance of progenitor cell fate in the anterior SHF essential for OFT separation, and why receptor dysfunction is a novel cause of conotruncal malformation.

Sortilin regulates sorting and secretion of Sonic hedgehog.

Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.

Endocytic receptor LRP2/megalin-of holoprosencephaly and renal Fanconi syndrome.

Megalin (or LRP2) is an endocytic receptor that plays a central role in embryonic development and adult tissue homeostasis. Loss of this receptor in congenital or acquired diseases results in multiple organ dysfunctions, including forebrain malformation (holoprosencephaly) and renal reabsorption defects (renal Fanconi syndrome). Here, we describe current concepts of the mode of receptor action that include co-receptors and a repertoire of different ligands, and we discuss how these interactions govern functional integrity of the kidney and the brain, and cause disease when defective.

Impaired vitreous composition and retinal pigment epithelium function in the FoxG1::LRP2 myopic mice.

High myopia (HM) is one of the main causes of visual impairment and blindness all over the world and an unsolved medical problem. Persons with HM are predisposed to other eye pathologies such as retinal detachment, myopic retinopathy or glaucomatous optic neuropathy, complications that may at least partly result from the extensive liquefaction of the myopic vitreous gel. To identify the involvement of the liquid vitreous in the pathogenesis of HM we here analyzed the vitreous of the recently described highly myopic low density lipoprotein receptor-related protein 2 (Lrp2)-deficient eyes. Whereas the gel-like fraction was not apparently modified, the volume of the liquid vitreous fraction (LVF) was much higher in the myopic eyes. Biochemical and proteome analysis of the LVF revealed several modifications including a marked decrease of potassium, sodium and chloride, of proteins involved in ocular tissue homeostasis and repair as well as of ADP-ribosylation factor 4 (ARF4), a protein possibly involved in LRP2 trafficking. A small number of proteins, mainly comprising known LRP2 ligands or proteins of the inflammatory response, were over expressed in the mutants. Moreover the morphology of the LRP2-deficient retinal pigment epithelium (RPE) cells was affected and the expression of ARF4 as well as of proteins involved in degradative endocytosis was strongly reduced. Our results support the idea that impairment of the RPE structure and most likely endocytic function may contribute to the vitreal modifications and pathogenesis of HM.

LRP2, an auxiliary receptor that controls sonic hedgehog signaling in development and disease.

To fulfill their multiple roles in organ development and adult tissue homeostasis, hedgehog (HH) morphogens act through their receptor Patched (PTCH) on target cells. However, HH actions also require HH binding proteins, auxiliary cell surface receptors that agonize or antagonize morphogen signaling in a context-dependent manner. Here, we discuss recent findings on the LDL receptor-related protein 2 (LRP2), an exemplary HH binding protein that modulates sonic hedgehog activities in stem and progenitor cell niches in embryonic and adult tissues. LRP2 functions are crucial for developmental processes in a number of tissues, including the brain, the eye, and the heart, and defects in this receptor pathway are the cause of devastating congenital diseases in humans. Developmental Dynamics 245:569-579, 2016. © 2016 Wiley Periodicals, Inc.

Endocytic receptor-mediated control of morphogen signaling.

Receptor-mediated endocytosis provides a mechanism by which cells take up signaling molecules from the extracellular space. Recent studies have shown that one class of endocytic receptors, the low-density lipoprotein receptor-related proteins (LRPs), is of particular relevance for embryonic development. In this Primer, we describe how LRPs constitute central pathways that modulate morphogen presentation to target tissues and cellular signal reception, and how LRP dysfunction leads to developmental disturbances in many species.

Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion.

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na(+),K(+)-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.

LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche.

The microenvironment of growth factors in the subependymal zone (SEZ) of the adult brain provides the instructive milieu for neurogenesis to proceed in this germinal niche. In particular, tight regulation of bone morphogenetic protein (BMP) signaling is essential to balance proliferative and non-proliferative cell fate specification. However, the regulatory pathways that control BMP signaling in the SEZ are still poorly defined. We demonstrate that LRP2, a clearance receptor for BMP4 is specifically expressed in ependymal cells of the lateral ventricles in the adult brain. Intriguingly, expression is restricted to the ependyma that faces the stem cell niche. Expression is not seen in ependyma elsewhere in the lateral ventricles or in the dentate gyrus, the second major neurogenic zone of the adult brain. We further show that lack of LRP2 expression in adult mice results in impaired proliferation of neural precursor cells in the SEZ resulting in decreased numbers of neuroblasts reaching the olfactory bulb. Reduced neurogenesis coincides with increased BMP4 expression and enhanced activation of downstream mediators phospho-SMAD1/5/8 and ID3 in the stem cell niche. Our findings suggest a novel mechanism whereby LRP2-mediated catabolism of BMP4 in the ependyma modulates the microenvironment of the SEZ and enables adult neurogenesis to proceed.

Cyclin M2 (CNNM2) knockout mice show mild hypomagnesaemia and developmental defects.

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/- mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2-/- pups were born alive. The Cnnm2-/- pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/- mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/- mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/- mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

The soluble intracellular domain of megalin does not affect renal proximal tubular function in vivo.

Megalin-mediated endocytic uptake constitutes the main pathway for clearance of plasma proteins from the glomerular filtrate in proximal tubules. Little is known, however, about mechanisms that control megalin expression and activity in the kidney. A widely discussed hypothesis states that upon ligand binding a regulated intramembrane proteolysis releases the cytosolic domain of megalin and this fragment subsequently modulates megalin gene transcription. Here, we tested this by generating a mouse model that co-expressed both the soluble intracellular domain and full-length megalin. Despite pronounced synthesis in the proximal tubules, the soluble intracellular domain failed to exert distinct effects on renal proximal tubular function, including megalin expression, endocytic retrieval of proteins, or global renal gene transcription. Hence, our study argues that the soluble intracellular domain does not have a role in regulating the activity of megalin in the kidney.

Cdon mutation and fetal alcohol converge on Nodal signaling in a mouse model of holoprosencephaly.

Holoprosencephaly (HPE), a defect in midline patterning of the forebrain and midface, arises ~1 in 250 conceptions. It is associated with predisposing mutations in the Nodal and Hedgehog (HH) pathways, with penetrance and expressivity graded by genetic and environmental modifiers, via poorly understood mechanisms. CDON is a multifunctional co-receptor, including for the HH pathway. In mice, Cdon mutation synergizes with fetal alcohol exposure, producing HPE phenotypes closely resembling those seen in humans. We report here that, unexpectedly, Nodal signaling is a major point of synergistic interaction between Cdon mutation and fetal alcohol. Window-of-sensitivity, genetic, and in vitro findings are consistent with a model whereby brief exposure of Cdon mutant embryos to ethanol during gastrulation transiently and partially inhibits Nodal pathway activity, with consequent effects on midline patterning. These results illuminate mechanisms of gene-environment interaction in a multifactorial model of a common birth defect.

A common birth defect known as holoprosencephaly affects how the brain and face of a fetus develop in the womb. In many cases, the condition is so severe that the fetus dies before, or shortly after, birth. Mutations in certain genes that control how the fetus develops are associated with holoprosencephaly. For example, mutations in components of the Hedgehog and Nodal signaling pathways, which transmit information that help cells to become specialized, increase the risk that a fetus will develop holoprosencephaly. Environmental factors, such as exposure to alcohol in the womb, are also thought to contribute to this condition. A gene known as Cdon is a component of the Hedgehog signaling pathway. In 2012, a team of researchers reported that mice with a mutation in the Cdon gene exposed to alcohol in the womb develop symptoms similar to holoprosencephaly in humans. Here, Hong et al. ­ including some of the researchers involved in the previous work ­ set out to understand how Cdon and alcohol work together to cause holoprosencephaly in the mutant mice. First, the team exposed pregnant mice to alcohol at different times during gestation to find out when their young were sensitive to developing holoprosencephaly. This showed that the young mice were most sensitive in early pregnancy when the Nodal pathway was active in their growing bodies. Further experiments found that alcohol and mutations in Cdon change Nodal signaling in cells. Together, these findings demonstrate that exposure to alcohol in the womb works together with the mutant form of Cdon via the Nodal signaling pathway, rather than the Hedgehog pathway, to cause holoprosencephaly in mice. The causes of many common birth defects are complex and difficult to distinguish at the level of individual cases. The work of Hong et al. illuminates how multiple risk factors during pregnancy, which may not create any problems on their own, may work together to produce birth defects in the fetus. The findings also offer new ways to understand how exposure to alcohol in the womb affects the fetus. Ultimately, understanding how birth defects form could lead to new strategies to prevent them in the future.

LRP2 Acts as SHH Clearance Receptor to Protect the Retinal Margin from Mitogenic Stimuli.

During forebrain development, LRP2 promotes morphogen signaling as an auxiliary SHH receptor. However, in the developing retina, LRP2 assumes the opposing function, mediating endocytic clearance of SHH and antagonizing morphogen action. LRP2-mediated clearance prevents spread of SHH activity from the central retina into the retinal margin to protect quiescent progenitor cells in this niche from mitogenic stimuli. Loss of LRP2 in mice increases the sensitivity of the retinal margin for SHH, causing expansion of the retinal progenitor cell pool and hyperproliferation of this tissue. Our findings document the ability of LRP2 to act, in a context-dependent manner, as activator or inhibitor of the SHH pathway. Our current findings uncovered LRP2 activity as the molecular mechanism imposing quiescence of the retinal margin in the mammalian eye and suggest SHH-induced proliferation of the retinal margin as cause of the large eye phenotype observed in mouse models and patients with LRP2 defects.

Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia.

Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM) is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS) and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5) and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM.

LRP2 is an auxiliary SHH receptor required to condition the forebrain ventral midline for inductive signals.

Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM), a major forebrain organizer. We identified LRP2, a member of the LDL receptor gene family, as a component of the SHH signaling machinery in the RDVM. LRP2 acts as an apical SHH-binding protein that sequesters SHH in its target field and controls internalization and cellular trafficking of SHH/patched 1 complexes. Lack of LRP2 in mice and in cephalic explants results in failure to respond to SHH, despite functional expression of patched 1 and smoothened, whereas overexpression of LRP2 variants in cells increases SHH signaling capacity. Our data identify a critical role for LRP2 in SHH signaling and reveal the molecular mechanism underlying forebrain anomalies in mice and patients with Lrp2 defects.