CERT1 mutations perturb human development by disrupting sphingolipid homeostasis

C. Gehin; M. A. Lone; W. Lee; L. Capolupo; S. Ho et al. 

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS

S. C. David; O. Vadas; I. Glas; A. Schaub; B. Luo et al. 

Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Protein changes were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future.

Circadian organization of lipid landscape is perturbed in type 2 diabetic patients

F. Sinturel; S. Chera; M. -C. Brulhart-Meynet; J. P. Montoya; D. J. Stenvers et al. 

Lipid homeostasis in humans follows a diurnal pattern in muscle and pancreatic islets, altered upon metabolic dysregulation. We employ tandem and liquid-chromatography mass spectrometry to investigate daily regulation of lipid metabolism in subcutaneous white adipose tissue (SAT) and serum of type 2 diabetic (T2D) and non-diabetic (ND) human volunteers (n = 12). Around 8% of z440 lipid metabolites exhibit diurnal rhythmicity in serum and SAT from ND and T2D subjects. The spectrum of rhythmic lipids differs between ND and T2D individuals, with the most substantial changes observed early morning, as confirmed by lipidomics in an independent cohort of ND and T2D subjects (n = 32) conducted at a single morning time point. Strikingly, metabolites identified as daily rhythmic in both serum and SAT from T2D subjects exhibit phase differences. Our study reveals massive temporal and tissue-specific alterations of human lipid homeostasis in T2D, providing essential clues for the development of lipid biomarkers in a temporal manner.

Sphingolipids control dermal fibroblast heterogeneity

L. Capolupo; I. Khven; A. R. Lederer; L. Mazzeo; G. Glousker et al. 

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains

M. U. Anwar; O. A. Sergeeva; L. Abrami; F. S. Mesquita; I. Lukonin et al. 

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regula-tory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligo-merization at the plasma membrane of human cells, an essential step dependent on localization to choles-terol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the dis-covery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3

R. Rizzo; D. Russo; K. Kurokawa; P. Sahu; B. Lombardi et al. 

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

P. Pothukuchi; I. Agliarulo; M. Pirozzi; R. Rizzo; D. Russo et al. 

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

F. S. Mesquita; L. Abrami; O. Sergeeva; P. Turelli; E. Qing et al. 

SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and

Sphingolipid metabolism and signaling: embracing diversity

C. J. Clarke; G. D’Angelo; L. C. Silva 

Glycosphingolipid metabolic reprogramming drives neural differentiation

D. Russo; F. Della Ragione; R. Rizzo; E. Sugiyama; F. Scalabrì et al. 

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.

Role and Function of Sphingomyelin Biosynthesis in the Development of Cancer

G. D’Angelo; S. Moorthi; C. Luberto 

Sphingomyelin (SM) biosynthesis represents a complex, finely regulated process, mostly occurring in vertebrates. It is intimately linked to lipid transport and it is ultimately carried out by two enzymes, SM synthase 1 and 2, selectively localized in the Golgi and plasma membrane. In the course of the SM biosynthetic reaction, various lipids are metabolized. Because these lipids have both structural and signaling functions, the SM biosynthetic process has the potential to affect diverse important cellular processes (such as cell proliferation, cell survival, and migration). Thus defects in SM biosynthesis might directly or indirectly impact the normal physiology of the cell and eventually of the organism. In this chapter, we will focus on evidence supporting a role for SM biosynthesis in specific cellular functions and how its dysregulation can affect neoplastic transformation.

Sphingolipid metabolic flow controls phosphoinositide turnover at the trans ‐Golgi network

G. D’Angelo; S. Capasso; L. Sticco; R. Rizzo; M. Pirozzi et al. 

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.

Phosphatidylinositol-4-phosphate: The Golgi and beyond

M. A. De Matteis; C. Wilson; G. D’Angelo 

Initially identified as a key phosphoinositide that controls membrane trafficking at the Golgi complex, phosphatidylinositol-4-phosphate (PI4P) has emerged as a key molecule in the regulation of a diverse array of cellular functions. In this review we will discuss selected examples of the findings that in the last few years have significantly increased our awareness of the regulation and roles of PI4P in the Golgi complex and beyond. We will also highlight the role of PI4P in infection and cancer. We believe that, with the increasing number of regulators and effectors of PI4P identified, the time is ripe for a more integrated approach of study. A first step in this direction is the delineation of PI4P-centered molecular networks that we provide using data from low and high throughput studies in yeast and mammals.

Glycosphingolipids: synthesis and functions

G. D’Angelo; S. Capasso; L. Sticco; D. Russo 

Glycosphingolipids (GSLs) comprise a heterogeneous group of membrane lipids formed by a ceramide backbone covalently linked to a glycan moiety. Hundreds of different glycans can be linked to tens of different ceramide molecules, giving rise to an astonishing variety of structurally different compounds, each of which has the potential for a specific biological function. GSLs have been suggested to modulate membrane‐protein function and to contribute to cell–cell communication. Although GSLs are dispensable for cellular life, they are indeed collectively required for the development of multicellular organisms, and are thus considered to be key molecules in ‘cell sociology’. Consequently, the GSL make‐up of individual cells is highly dynamic and is strictly linked to the cellular developmental and environmental state. In the present review, we discuss some of the available knowledge, open questions and future perspectives relating to the study of GSL biology.

Connecting vesicular transport with lipid synthesis: FAPP2

G. D’Angelo; L. R. Rega; M. A. De Matteis 

Next to the protein-based machineries composed of small G-proteins, coat complexes, SNAREs and tethering factors, the lipid-based machineries are emerging as important players in membrane trafficking. As a component of these machineries, lipid transfer proteins have recently attracted the attention of cell biologists for their involvement in trafficking along different segments of the secretory pathway. Among these, the four-phosphate adaptor protein 2 (FAPP2) was discovered as a protein that localizes dynamically with the trans-Golgi network and regulates the transport of proteins from the Golgi complex to the cell surface. Later studies have highlighted a role for FAPP2 as lipid transfer protein involved in glycosphingolipid metabolism at the Golgi complex. Here we discuss the available evidence on the function of FAPP2 in both membrane trafficking and lipid metabolism and propose a mechanism of action of FAPP2 that integrates its activities in membrane trafficking and in lipid transfer. This article is part of a Special Issue entitled Lipids and Vesicular Transport.

The Golgi apparatus: an organelle with multiple complex functions

C. Wilson; R. Venditti; L. Rega; A. Colanzi; G. D’Angelo et al. 

Remarkable advances have been made during the last few decades in defining the organizational principles of the secretory pathway. The Golgi complex in particular has attracted special attention due to its central position in the pathway, as well as for its fascinating and complex structure. Analytical studies of this organelle have produced significant advances in our understanding of its function, although some aspects still seem to elude our comprehension. In more recent years a level of complexity surrounding this organelle has emerged with the discovery that the Golgi complex is involved in cellular processes other than the ‘classical’ trafficking and biosynthetic pathways. The resulting picture is that the Golgi complex can be considered as a cellular headquarters where cargo sorting/processing, basic metabolism, signalling and cell-fate decisional processes converge.

Identification of microRNA-regulated gene networks by expression analysis of target genes

V. A. Gennarino; G. D’Angelo; G. Dharmalingam; S. Fernandez; G. Russolillo et al. 

MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs.

GRASP65 and GRASP55 Sequentially Promote the Transport of C-terminal Valine-bearing Cargos to and through the Golgi Complex

G. D’Angelo; L. Prencipe; L. Iodice; G. Beznoussenko; M. Savarese et al. 

The Golgi matrix proteins GRASP65 and GRASP55 have recognized roles in maintaining the architecture of the Golgi complex, in mitotic progression and in unconventional protein secretion whereas, surprisingly, they have been shown to be dispensable for the transport of commonly used reporter cargo proteins along the secretory pathway. However, it is becoming increasingly clear that many trafficking machineries operate in a cargo-specific manner, thus we have investigated whether GRASPs may control the trafficking of selected classes of cargo. We have taken into consideration the C-terminal valine-bearing receptors CD8alpha and Frizzled4 that we show bind directly to the PSD95-DlgA-zo-1 (PDZ) domains of GRASP65 and GRASP55. We demonstrate that both GRASPs are needed sequentially for the efficient transport to and through the Golgi complex of these receptors, thus highlighting a novel role for the GRASPs in membrane trafficking. Our results open new perspectives for our understanding of the regulation of surface expression of a class of membrane proteins, and suggests the causal mechanisms of a dominant form of autosomal human familial exudative vitreoretinopathy that arises from the Frizzled4 mutation involving its C-terminal valine.

Function and dysfunction of the PI system in membrane trafficking

M. Vicinanza; G. D’Angelo; A. Di Campli; M. A. De Matteis 

The role of the phosphoinositides at the Golgi complex

M. A. De Matteis; G. D’Angelo 

Eukaryotic cells are organized into a complex system of subcompartments, each with its distinct protein and lipid composition. A continuous flux of membranes crosses these compartments, and in some cases direct connections exist between the different organelles. It is thus surprising that they can maintain their individual identities. Small GTPases and the phosphoinositides have emerged as the key regulators in the maintenance of the identity of the Golgi complex. This property is due to their ability to act either alone or, more often, in combination, as cues directing and controlling the recruitment of proteins that possess phosphoinositide-binding domains. Among these many proteins there are the lipid transfer proteins, which can transfer ceramide, oxysterol, cholesterol and possibly glucosylceramide. By regulating these lipid transfer proteins in this way, this binomial combination of the small GTPases and the phosphoinositides acquires a further important role: control of the synthesis and/or distribution of other important integral constituents of cell organelles, such as the sphingolipids and cholesterol. This role is particularly relevant at the level of the Golgi complex, a key organelle in the biosynthesis, transport and sorting of both lipids and proteins that is located at the intersection of the secretory and endocytic pathways.

Lipid-transfer proteins in membrane trafficking at the Golgi complex

M. A. De Matteis; A. Di Campli; G. D’Angelo 

The Golgi complex (GC) represents the central junction for membrane trafficking. Protein and lipid cargoes continuously move through the GC in both anterograde and retrograde directions, departing to and arriving from diverse destinations within the cell. Nevertheless, the GC is able to maintain its identity and strict compartmentalisation, having a different composition in terms of protein and lipid content compared to other organelles. The discovery of coat protein complexes and the elucidation of their role in sorting cargo proteins into specific transport carriers have provided a partial answer to this phenomenon. However, it is more difficult to understand how relatively small and diffusible molecules like lipids can be concentrated in or excluded from specific subcellular compartments. The discovery of lipid-transfer proteins operating in the secretory pathway and specifically at the GC has shed light on one possible way in which this lipid compartmentalisation can be accomplished. The correct lipid distribution along the secretory pathway is of crucial importance for cargo protein sorting and secretion. This review focuses on what is now known about the putative and effective lipid-transfer proteins at the GC, and on how they affect the function and structure of the GC itself.

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide

G. D’Angelo; E. Polishchuk; G. D. Tullio; M. Santoro; A. D. Campli et al. 

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.