Mechanisms and functions of protein S-acylation.

Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is a reversible post-translational modification that involves the attachment of a fatty acid to a protein. Maintenance of the equilibrium between protein S-acylation and deacylation has demonstrated profound effects on various cellular processes, including innate immunity, inflammation, glucose metabolism and fat metabolism, as well as on brain and heart function. This Review provides an overview of current understanding of S-acylation and deacylation enzymes, their spatiotemporal regulation by sophisticated multilayered mechanisms, and their influence on protein function, cellular processes and physiological pathways. Furthermore, we examine how disruptions in protein S-acylation are associated with a broad spectrum of diseases from cancer to autoinflammatory disorders and neurological conditions.

Kicking Out Pathogens in Exosomes.

Host-pathogen interactions involve a series of attacks and counter-attacks. Miao et al. show that, although some invading bacteria can take shelter in lysosomes by neutralizing their pH, this respite is temporary, as host cells can expel them in exosomes.

Targeting STING with covalent small-molecule inhibitors.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains.

Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux.

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell's refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells' division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.

Anthrax toxin requires ZDHHC5-mediated palmitoylation of its surface-processing host enzymes.

The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell.

Mammalian membrane trafficking as seen through the lens of bacterial toxins.

A fundamental question of eukaryotic cell biology is how membrane organelles are organised and interact with each other. Cell biologists address these questions by characterising the structural features of membrane compartments and the mechanisms that coordinate their exchange. To do so, they must rely on variety of cargo molecules and treatments that enable targeted perturbation, localisation, and labelling of specific compartments. In this context, bacterial toxins emerged in cell biology as paradigm shifting molecules that enabled scientists to not only study them from the side of bacterial infection but also from the side of the mammalian host. Their selectivity, potency, and versatility made them exquisite tools for uncovering much of our current understanding of membrane trafficking mechanisms. Here, we will follow the steps that lead toxins until their intracellular targets, highlighting how specific events helped us comprehend membrane trafficking and establish the fundamentals of various cellular organelles and processes. Bacterial toxins will continue to guide us in answering crucial questions in cellular biology while also acting as probes for new technologies and applications.

Calnexin controls the STAT3-mediated transcriptional response to EGF.

Calnexin is a well-characterized transmembrane chaperone involved in the folding of newly synthesized glycoproteins in the lumen of the endoplasmic reticulum (ER). Here, we reveal a previously unrecognized function of calnexin in regulating the transcriptional response downstream of epidermal growth factor receptor (EGF), the product of a well-known human oncogene. We find that cell stimulation with EGF leads to the caspase-8-dependent cleavage of the calnexin cytoplasmic domain, preferentially at ER-mitochondria interaction sites. The released fragment translocates into the nucleus, binds to PIAS3--a natural inhibitor of activated STAT3--and, thus, acts as an enhancer of the STAT3-mediated transcriptional response to EGF. Also, we reveal the unsuspected capacity of calnexin to sense ER stress and, in response, prevent the EGF-induced processing of its cytosolic domain. Thus, cells integrate the health status of the ER to determine the amplitude of their response to EGF.

Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20.

Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain-containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by Acyl-RAC and confirmed by 3H-palmitate labeling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co-localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the HA-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs.

The molecular era of protein S-acylation: spotlight on structure, mechanisms, and dynamics.

S-Acylation (commonly referred to as S-palmitoylation) is a post-translational modification consisting in the covalent attachment of an acyl chain to a cysteine residue of the target protein. The lability of the resulting thioester bond gives S-acylation an essential characteristic: its reversibility. S-acylation dynamically regulates different aspects in the life of a protein (including stability, localization, interactome, and function) and, thus, plays critical roles in cellular physiology. For long, the reversibility of S-acylation has been neglected and thereby its potential as a regulatory mechanism for protein function undervalued. Thanks to technological advances, the field has now entered its golden era. A great diversity of interesting targets is being identified, the physio-pathological importance of the modification is starting to be revealed, structural information on the enzymes is becoming available, and the regulatory dynamics are gradually being understood. Here we will review the most recent literature in the S-acylation field, with a special focus on the molecular aspects of the modification, its regulation, and its consequences.

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.

Damage of eukaryotic cells by the pore-forming toxin sticholysin II: Consequences of the potassium efflux.

Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin.

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.

Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.

Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.

The dark sides of capillary morphogenesis gene 2.

Capillary morphogenesis gene 2 (CMG2) is a type I membrane protein involved in the homeostasis of the extracellular matrix. While it shares interesting similarities with integrins, its exact molecular role is unknown. The interest and knowledge about CMG2 largely stems from the fact that it is involved in two diseases, one infectious and one genetic. CMG2 is the main receptor of the anthrax toxin, and knocking out this gene in mice renders them insensitive to infection with Bacillus anthracis spores. On the other hand, mutations in CMG2 lead to a rare but severe autosomal recessive disorder in humans called Hyaline Fibromatosis Syndrome (HFS). We will here review what is known about the structure of CMG2 and its ability to mediate anthrax toxin entry into cell. We will then describe the limited knowledge available concerning the physiological role of CMG2. Finally, we will describe HFS and the consequences of HFS-associated mutations in CMG2 at the molecular and cellular level.

Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis.

Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus α-toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a high-molecular-weight complex in α-toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis.

The SwissLipids knowledgebase for lipid biology.

MOTIVATION: Lipids are a large and diverse group of biological molecules with roles in membrane formation, energy storage and signaling. Cellular lipidomes may contain tens of thousands of structures, a staggering degree of complexity whose significance is not yet fully understood. High-throughput mass spectrometry-based platforms provide a means to study this complexity, but the interpretation of lipidomic data and its integration with prior knowledge of lipid biology suffers from a lack of appropriate tools to manage the data and extract knowledge from it. RESULTS: To facilitate the description and exploration of lipidomic data and its integration with prior biological knowledge, we have developed a knowledge resource for lipids and their biology-SwissLipids. SwissLipids provides curated knowledge of lipid structures and metabolism which is used to generate an in silico library of feasible lipid structures. These are arranged in a hierarchical classification that links mass spectrometry analytical outputs to all possible lipid structures, metabolic reactions and enzymes. SwissLipids provides a reference namespace for lipidomic data publication, data exploration and hypothesis generation. The current version of SwissLipids includes over 244 000 known and theoretically possible lipid structures, over 800 proteins, and curated links to published knowledge from over 620 peer-reviewed publications. We are continually updating the SwissLipids hierarchy with new lipid categories and new expert curated knowledge. AVAILABILITY: SwissLipids is freely available at http://www.swisslipids.org/. CONTACT: alan.bridge@isb-sib.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

How many lives does CLIMP-63 have?

In 1995, in the Biochemical Society Transactions, Mundy published the first review on CLIMP-63 (cytoskeleton-linking membrane protein 63) or CKPA4 (cytoskeleton-associated protein 4), initially just p63 [1]. Here we review the following 20 years of research on this still mysterious protein. CLIMP-63 is a type II transmembrane protein, the cytosolic domain of which has the capacity to bind microtubules whereas the luminal domain can form homo-oligomeric complexes, not only with neighbouring molecules but also, in trans, with CLIMP-63 molecules on the other side of the endoplasmic reticulum (ER) lumen, thus promoting the formation of ER sheets. CLIMP-63 however also appears to have a life at the cell surface where it acts as a ligand-activated receptor. The still rudimentary information of how CLIMP-63 fulfills these different roles, what these are exactly and how post-translational modifications control them, will be discussed.

Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism.

Aerolysin is the founding member of a superfamily of ß-pore-forming toxins whose pore structure is unknown. We have combined X-ray crystallography, cryo-EM, molecular dynamics and computational modeling to determine the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extracted from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochemical data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane ß-barrel.