Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

The C-terminal tail of CSNAP attenuates the CSN complex.

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

Blocking an epitope of misfolded SOD1 ameliorates disease phenotype in a model of amyotrophic lateral sclerosis.

The current strategies to mitigate the toxicity of misfolded superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis via blocking SOD1 expression in the CNS are indiscriminative for misfolded and intact proteins, and as such, entail a risk of depriving CNS cells of their essential antioxidant potential. As an alternative approach to neutralize misfolded and spare unaffected SOD1 species, we developed scFv-SE21 antibody that blocks the ß6/ß7 loop epitope exposed exclusively in misfolded SOD1. The ß6/ß7 loop epitope has previously been proposed to initiate amyloid-like aggregation of misfolded SOD1 and mediate its prion-like activity. The adeno-associated virus-mediated expression of scFv-SE21 in the CNS of hSOD1G37R mice rescued spinal motor neurons, reduced the accumulation of misfolded SOD1, decreased gliosis and thus delayed disease onset and extended survival by 90 days. The results provide evidence for the role of the exposed ß6/ß7 loop epitope in the mechanism of neurotoxic gain-of-function of misfolded SOD1 and open avenues for the development of mechanism-based anti-SOD1 therapeutics, whose selective targeting of misfolded SOD1 species may entail a reduced risk of collateral oxidative damage to the CNS.

Stable Mammalian Serum Albumins Designed for Bacterial Expression.

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.

Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction.

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 µM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1ß (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy.Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Intestinal mucin is a chaperone of multivalent copper.

Mucus protects the epithelial cells of the digestive and respiratory tracts from pathogens and other hazards. Progress in determining the molecular mechanisms of mucus barrier function has been limited by the lack of high-resolution structural information on mucins, the giant, secreted, gel-forming glycoproteins that are the major constituents of mucus. Here, we report how mucin structures we determined enabled the discovery of an unanticipated protective role of mucus: managing the toxic transition metal copper. Using two juxtaposed copper binding sites, one for Cu2+ and the other for Cu1+, the intestinal mucin, MUC2, prevents copper toxicity by blocking futile redox cycling and the squandering of dietary antioxidants, while nevertheless permitting uptake of this important trace metal into cells. These findings emphasize the value of molecular structure in advancing mucosal biology, while introducing mucins, produced in massive quantities to guard extensive mucosal surfaces, as extracellular copper chaperones.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories.

The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.

Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.

Infection cycles of large DNA viruses: emerging themes and underlying questions.

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these 'nuclear-like' organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the 'stargate' portal that is used for genome release. Such a 'division of labor' is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed.

Delayed development induced by toxicity to the host can be inherited by a bacterial-dependent, transgenerational effect.

Commensal gut bacteria in many species including flies are integral part of their host, and are known to influence its development and homeostasis within generation. Here we report an unexpected impact of host-microbe interactions, which mediates multi-generational, non-Mendelian inheritance of a stress-induced phenotype. We have previously shown that exposure of fly larvae to G418 antibiotic induces transgenerationally heritable phenotypes, including a delay in larval development, gene induction in the gut and morphological changes. We now show that G418 selectively depletes commensal Acetobacter species and that this depletion explains the heritable delay, but not the inheritance of the other phenotypes. Notably, the inheritance of the delay was mediated by a surprising trans-generational effect. Specifically, bacterial removal from F1 embryos did not induce significant delay in F1 larvae, but nonetheless led to a considerable delay in F2. This effect maintains a delay induced by bacterial-independent G418 toxicity to the host. In line with these findings, reintroduction of isolated Acetobacter species prevented the inheritance of the delay. We further show that this prevention is partly mediated by vitamin B2 (Riboflavin) produced by these bacteria; exogenous Riboflavin led to partial prevention and inhibition of Riboflavin synthesis compromised the ability of the bacteria to prevent the inheritance. These results identify host-microbe interactions as a hitherto unrecognized factor capable of mediating non-Mendelian inheritance of a stress-induced phenotype.

Stress-induced condensation of bacterial genomes results in re-pairing of sister chromosomes: implications for double strand DNA break repair.

Genome condensation is increasingly recognized as a generic stress response in bacteria. To better understand the physiological implications of this response, we used fluorescent markers to locate specific sites on Escherichia coli chromosomes following exposure to cytotoxic stress. We find that stress-induced condensation proceeds through a nonrandom, zipper-like convergence of sister chromosomes, which is proposed to rely on the recently demonstrated intrinsic ability of identical double-stranded DNA molecules to specifically identify each other. We further show that this convergence culminates in spatial proximity of homologous sites throughout chromosome arms. We suggest that the resulting apposition of homologous sites can explain how repair of double strand DNA breaks might occur in a mechanism that is independent of the widely accepted yet physiologically improbable genome-wide search for homologous templates. We claim that by inducing genome condensation and orderly convergence of sister chromosomes, diverse stress conditions prime bacteria to effectively cope with severe DNA lesions such as double strand DNA breaks.

Epigenetically heritable alteration of fly development in response to toxic challenge.

Developing organisms have evolved a wide range of mechanisms for coping with recurrent environmental challenges. How they cope with rare or unforeseen challenges is, however, unclear as are the implications to their unchallenged offspring. Here, we investigate these questions by confronting the development of the fly, D. melanogaster, with artificial tissue distributions of toxic stress that are not expected to occur during fly development. We show that under a wide range of toxic scenarios, this challenge can lead to modified development that may coincide with increased tolerance to an otherwise lethal condition. Part of this response was mediated by suppression of Polycomb group genes, which in turn leads to derepression of developmental regulators and their expression in new domains. Importantly, some of the developmental alterations were epigenetically inherited by subsequent generations of unchallenged offspring. These results show that the environment can induce alternative patterns of development that are stable across multiple generations.

Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging.

One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus-HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine-phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

Conformational stability and membrane interaction of the full-length ectodomain of HIV-1 gp41: implication for mode of action.

Membrane fusion between the human immunodeficiency virus (HIV) and the target cell plasma membrane is correlated with conformational changes in the HIV gp41 glycoprotein, which include an early exposed conformation (prehairpin) and a late low energy six helix bundle (SHB) conformation also termed hairpin. Peptides resembling regions from the exposed prehairpin have been previously studied for their interaction with membranes. Here we report on the expression, purification, SHB stability, and membrane interaction of the full-length ectodomain of the HIV gp41 and its two deletion mutants, all in their SHB-folded state. The interaction of the proteins with zwitterionic and negatively charged membranes was examined by using various biophysical methods including circular dichroism spectroscopy, differential scanning calorimetry, lipid mixing of large unilamellar vesicles, and atomic force microscopy (AFM). All experiments were done in an acidic environment in which the protein remains in its soluble trimeric state. The data reveal that all three proteins fold into a stable coiled-coil core in aqueous solution and retain a stable helical fold with reduced coiled-coil characteristics in a zwitterionic and negatively charged membrane mimetic environment. Furthermore, in contrast with the extended exposed N-terminal domain, the folded gp41 ectodomain does not induce lipid mixing of zwitterionic membranes. However, it disrupts and induces lipid mixing of negatively charged phospholipid membranes (approximately 100-fold more effective than fusion peptide alone), which are known to be expressed more in HIV-1-infected T cells or macrophages. The results support the emerging model in which one of the roles of gp41 folding into the SHB conformation is to slow down membrane disruption effects induced by early exposed gp41. However, it can further affect membrane morphology once exposed to negatively charged membranes during late stages.

Mode of membrane interaction and fusogenic properties of a de novo transmembrane model peptide depend on the length of the hydrophobic core.

Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.

C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin.

Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.

Structural analysis of the interaction between the SNARE Tlg1 and Vps51.

Membrane fusion in cells involves the interaction of SNARE proteins on apposing membranes. Formation of SNARE complexes is preceded by tethering events, and a number of protein complexes that are thought to mediate this have been identified. The VFT or GARP complex is required for endosome-Golgi traffic in yeast. It consists of four subunits, one of which, Vps51, has been shown to bind specifically to the SNARE Tlg1, which participates in the same fusion event. We have determined the structure of the N-terminal domain of Tlg1 bound to a peptide from the N terminus of Vps51. Binding depends mainly on residues 18-30 of Vps51. These form a short helix which lies in a conserved groove in the three-helix bundle formed by Tlg1. Surprisingly, although both Vps51 and Tlg1 are required for transport to the late Golgi from endosomes, removal of the Tlg1-binding sequences from Vps51 does not block such traffic in vivo. Thus, this particular interaction cannot be crucial to the process of vesicle docking or fusion.

TMF is a golgin that binds Rab6 and influences Golgi morphology.

BACKGROUND: Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association. Many are peripheral membrane proteins recruited by GTPases. Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved. RESULTS: We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes. The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor. We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6. Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks. CONCLUSION: We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells.