Redefining Hypo- and Hyper-Responding Phenotypes of CFTR Mutants for Understanding and Therapy

Mutations in CFTR cause misfolding and decreased or absent ion-channel function, resulting in the disease Cystic Fibrosis. Fortunately, a triple-modulator combination therapy (Trikafta) has been FDA-approved for 178 mutations, including all patients who have F508del on one allele. That so many CFTR mutants respond well to modulators developed for a single mutation is due to the nature of the folding process of this multidomain protein. We have addressed the question 'What characterizes the exceptions: the mutants that functionally respond either not or extremely well'. A functional response is the product of the number of CFTR molecules on the cell surface, open probability, and conductivity of the CFTR chloride channel. By combining biosynthetic radiolabeling with protease-susceptibility assays, we have followed CF-causing mutants during the early and late stages of folding in the presence and absence of modulators. Most CFTR mutants showed typical biochemical responses for each modulator, such as a TMD1 conformational change or an increase in (cell-surface) stability, regardless of a functional response. These modulators thus should still be considered for hypo-responder genotypes. Understanding both biochemical and functional phenotypes of outlier mutations will boost our insights into CFTR folding and misfolding, and lead to improved therapeutic strategies

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids

Instability of Acylcarnitines in Stored Dried Blood Spots:The Impact on Retrospective Analysis of Biomarkers for Inborn Errors of Metabolism

Stored dried blood spots (DBS) can provide valuable samples for the retrospective diagnosis of inborn errors of metabolism, and for validation studies for newborn blood spot screening programs. Acylcarnitine species are subject to degradation upon long-term storage at room temperature, but limited data are available on the stability in original samples and the impact on acylcarnitine ratios. We analysed complete acylcarnitine profiles by flow-injection tandem mass spectrometry in 598 anonymous DBS stored from 2013 to 2017, at +4 degrees C during the first year and thereafter at room temperature. The concentrations of C2-, C3-, C4-, C5-, C6-, C8-, C10:1-, C10-, C12:1-, C12-, C14:1-, C14-, C16:1-, C16-, C18:2-, C18:1-, C18-, C5OH+C4DC-, C18:1OH-, and C16DC-carnitine decreased significantly, whereas a positive trend was found for free carnitine. Only the C4/C8-, C8/C10-, C14:1/C10- and C14:1/C16-carnitine ratios appeared robust for the metabolite instability. The metabolite instability may provoke the wrong interpretation of test results in the case of retrospective studies and risk the inaccurate estimation of cut-off targets in validation studies when only stored control DBS are used. We recommend including control DBS in diagnostic, retrospective cohort studies, and, for validation studies, we recommend using fresh samples and repeatedly re-evaluating cut-off targets

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes

Improved Model-Data Agreement With Strongly Eddying Ocean Simulations in the Middle-Late Eocene

Model simulations of past climates are increasingly found to compare well with proxy data at a global scale, but regional discrepancies remain. A persistent issue in modeling past greenhouse climates has been the temperature difference between equatorial and (sub-)polar regions, which is typically much larger in simulations than proxy data suggest. Particularly in the Eocene, multiple temperature proxies suggest extreme warmth in the southwest Pacific Ocean, where model simulations consistently suggest temperate conditions. Here, we present new global ocean model simulations at 0.1° horizontal resolution for the middle-late Eocene. The eddies in the high-resolution model affect poleward heat transport and local time-mean flow in critical regions compared to the noneddying flow in the standard low-resolution simulations. As a result, the high-resolution simulations produce higher surface temperatures near Antarctica and lower surface temperatures near the equator compared to the low-resolution simulations, leading to better correspondence with proxy reconstructions. Crucially, the high-resolution simulations are also much more consistent with biogeographic patterns in endemic-Antarctic and low-latitude-derived plankton, and thus resolve the long-standing discrepancy of warm subpolar ocean temperatures and isolating polar gyre circulation. The results imply that strongly eddying model simulations are required to reconcile discrepancies between regional proxy data and models, and demonstrate the importance of accurate regional paleobathymetry for proxy-model comparisons

Long-term Phanerozoic global mean sea level: Insights from strontium isotope variations and estimates of continental glaciation

Global mean sea level is a key component within the fields of climate and oceanographic modelling in the Anthropocene. Hence, an improved understanding of eustatic sea level in deep time aids in our understanding of Earth's paleoclimate and may help predict future climatological and sea level changes. However, long-term eustatic sea level reconstructions are hampered because of ambiguity in stratigraphic interpretations of the rock record and limitations in plate tectonic modelling. Hence the amplitude and timescales of Phanerozoic eustasy remains poorly constrained. A novel, independent method from stratigraphic or plate modelling methods, based on estimating the effect of plate tectonics (i.e., mid-ocean ridge spreading) from the 87Sr/86Sr record led to a long-term eustatic sea level curve, but did not include glacio-eustatic drivers. Here, we incorporate changes in sea level resulting from variations in seawater volume from continental glaciations at time steps of 1 Myr. Based on a recent compilation of global average paleotemperature derived from δ18O data, paleo-Köppen zones and paleogeographic reconstructions, we estimate ice distribution on land and continental shelf margins. Ice thickness is calibrated with a recent paleoclimate model for the late Cenozoic icehouse, yielding an average ∼1.4 km thickness for land ice, ultimately providing global ice volume estimates. Eustatic sea level variations associated with long-term glaciations (>1 Myr) reach up to ∼90 m, similar to, and is at times dominant in amplitude over plate tectonic-derived eustasy. We superimpose the long-term sea level effects of land ice on the plate tectonically driven sea level record. This results in a Tectono-Glacio-Eustatic (TGE) curvefor which we describe the main long-term (>50 Myr) and residual trends in detail

MYO5B, STX3, and STXBP2 mutations reveal a common disease mechanism that unifies a subset of congenital diarrheal disorders:A mutation update

Microvillus inclusion disease (MVID) is a rare but fatal autosomal recessive congenital diarrheal disorder caused by MYO5B mutations. In 2013, we launched an open-access registry for MVID patients and their MYO5B mutations (www.mvid-central.org). Since then, additional unique MYO5B mutations have been identified in MVID patients, but also in non-MVID patients. Animal models have been generated that formally prove the causality between MYO5B and MVID. Importantly, mutations in two other genes, STXBP2 and STX3, have since been associated with variants of MVID, shedding new light on the pathogenesis of this congenital diarrheal disorder. Here, we review these additional genes and their mutations. Furthermore, we discuss recent data from cell studies that indicate that the three genes are functionally linked and, therefore, may constitute a common disease mechanism that unifies a subset of phenotypically linked congenital diarrheal disorders. We present new data based on patient material to support this. To congregate existing and future information on MVID geno-/phenotypes, we have updated and expanded the MVID registry to include all currently known MVID-associated gene mutations, their demonstrated or predicted functional consequences, and associated clinical information.</p