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

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 regulatory, 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 oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-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 discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications.

Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.

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.

Converging physiological roles of the anthrax toxin receptors.

The anthrax toxin receptors-capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8)-were identified almost 20 years ago, although few studies have moved beyond their roles as receptors for the anthrax toxins to address their physiological functions. In the last few years, insight into their endogenous roles has come from two rare diseases: hyaline fibromatosis syndrome, caused by mutations in CMG2, and growth retardation, alopecia, pseudo-anodontia, and optic atrophy (GAPO) syndrome, caused by loss-of-function mutations in TEM8. Although CMG2 and TEM8 are highly homologous at the protein level, the difference in disease symptoms points to variations in the physiological roles of the two anthrax receptors. Here, we focus on the similarities between these receptors in their ability to regulate extracellular matrix homeostasis, angiogenesis, cell migration, and skin elasticity. In this way, we shed light on how mutations in these two related proteins cause such seemingly different diseases and we highlight the existing knowledge gaps that could form the focus of future studies.

Co-expression of CCT subunits hints at TRiC assembly.

The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin) with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.

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.

Structure of the human TRiC/CCT Subunit 5 associated with hereditary sensory neuropathy.

The human chaperonin TRiC consists of eight non-identical subunits, and its protein-folding activity is critical for cellular health. Misfolded proteins are associated with many human diseases, such as amyloid diseases, cancer, and neuropathies, making TRiC a potential therapeutic target. A detailed structural understanding of its ATP-dependent folding mechanism and substrate recognition is therefore of great importance. Of particular health-related interest is the mutation Histidine 147 to Arginine (H147R) in human TRiC subunit 5 (CCT5), which has been associated with hereditary sensory neuropathy. In this paper, we describe the crystal structures of CCT5 and the CCT5-H147R mutant, which provide important structural information for this vital protein-folding machine in humans. This first X-ray crystallographic study of a single human CCT subunit in the context of a hexadecameric complex can be expanded in the future to the other 7 subunits that form the TRiC complex.

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.

Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography.

Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

Biochemical characterization of mutants in chaperonin proteins CCT4 and CCT5 associated with hereditary sensory neuropathy.

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate ß-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.

Group II archaeal chaperonin recognition of partially folded human γD-crystallin mutants.

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized ß-sheet lens protein human γD-crystallin (HγD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD-Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HγD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HγD-Crys mutants better than (wild-type) WT and refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm-Cpn does not recognize the features of HγD-Crys tested-paired aromatics, exposed domain interface, or destabilized core-but rather recognizes other features of the partially folded ß-sheet conformation that are absent or inaccessible in the native state of HγD-Crys.

Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro.

Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1-8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

Kinetic properties of human thymidylate synthase, an anticancer drug target.

We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2(')-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH(2)-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011.