Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes
Qi Xiao, Srujana S. Yadavalli, Shaodong Zhang, Samuel E. Sherman, Elodie Fiorin, Louise da Silva, Daniela A. Wilson, Daniel A. Hammer, Sabine André, Hans-Joachim Gabius, Michael L. Klein, Mark Goulian, and Virgil Percec
Cell surface determinants such as glycans, receptors, and adhesion molecules govern cell sociology in a complex manner. By forming cell-like hybrids of chemically programmable (glyco)dendrimersomes with bacterial membrane vesicles, evidence is obtained for the feasibility of combining chemical and biological surface design in one entity. Such tunable cell-like hybrids with custom-made combinations of surface epitopes and active receptors will likely find utility in dissecting the functionality of individual entities in complex networks and ultimately enable novel biomedical applications. (See pp. E1134–E1141.)
Tailoring nanoparticle designs to target cancer based on tumor pathophysiology
Edward A. Sykes, Qin Dai, Christopher D. Sarsons, Juan Chen, Jonathan V. Rocheleau, David M. Hwang, Gang Zheng, David T. Cramb, Kristina D. Rinker, and Warren C. W. Chan
Nanotechnology is a promising approach for improving cancer diagnosis and treatment with reduced side effects. A key question that has emerged is: What is the ideal nanoparticle size, shape, or surface chemistry for targeting tumors? Here, we show that tumor pathophysiology and volume can significantly impact nanoparticle targeting. This finding presents a paradigm shift in nanomedicine away from identifying and using a universal nanoparticle design for cancer detection and treatment. Rather, our results suggest that future clinicians will be capable of tailoring nanoparticle designs according to the patient's tumor characteristics. This concept of “personalized nanomedicine” was tested for detection of prostate tumors and was successfully demonstrated to improve nanoparticle targeting by over 50%. (See pp. E1142–E1151.)
Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR
Loes M. Stevers, Chan V. Lam, Seppe F. R. Leysen, Femke A. Meijer, Daphne S. van Scheppingen, Rens M. J. M. de Vries, Graeme W. Carlile, Lech G. Milroy, David Y. Thomas, Luc Brunsveld, and Christian Ottmann
It has been shown that 14-3-3 proteins increase trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane by binding to its regulatory (R) domain. This paper contains a detailed characterization of the 14-3-3/CFTR interaction, showing that multiple phosphorylated binding sites in the CFTR R-domain are necessary for significant binding with 14-3-3. We find that one of these binding sites serves as an anchor, while surrounding weaker sites enhance the interaction. Furthermore, we show the druggability of this interaction using natural-product fusicoccin-A, which stabilizes the 14-3-3/CFTR interaction by selectively modifying a weaker binding site. This mechanism of action can serve as a model for the development of new trafficking corrector molecules to treat cystic fibrosis. (See pp. E1152–E1161.)
Calcium can mobilize and activate myosin-VI
Christopher Batters, Dario Brack, Heike Ellrich, Beate Averbeck, and Claudia Veigel
The timing of motor protein activation is central to a broad range of cellular motile processes including endocytosis, cell division, and cancer cell migration. The cytoskeletal motor myosin-VI is involved in these processes and is the only myosin in the human genome shown to move toward the minus end of actin filaments. Using electron microscopy, fluorescence spectroscopy, and motility assays, we demonstrate that calcium is the cellular switch that directs the rearrangement of the motor from a dormant, inactive state at low calcium to a cargo-binding nonmotile state at high calcium. The return to low calcium generates either cargo-bound active motors that translocate to the center of the cell or refolded inactive motors ready for the next cellular calcium flux. (See pp. E1162–E1169.)
Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins
Logan R. Myler, Ignacio F. Gallardo, Yi Zhou, Fade Gong, Soo-Hyun Yang, Marc S. Wold, Kyle M. Miller, Tanya T. Paull, and Ilya J. Finkelstein
Exonuclease 1 (Exo1) is a conserved eukaryotic nuclease that participates in DNA repair and telomere maintenance. Here we use high-throughput single-molecule imaging to examine Exo1 activity on DNA and in the presence of single-stranded DNA binding proteins. We report that both human and yeast Exo1 are processive nucleases but are rapidly turned over by replication protein A (RPA). In the presence of RPA, Exo1 retains limited DNA-processing activity, albeit via a distributive binding mechanism. This rapid turnover by RPA can appear stimulatory or inhibitory in gel-based assays, clarifying conflicting results in the existing literature. RPA-depleted human cells show elevated Exo1 loading but reduced overall DNA resection, underscoring the many roles of RPA in regulating DNA resection in vivo. (See pp. E1170–E1179.)
70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria
Hiroshi Yamamoto, Daniela Wittek, Romi Gupta, Bo Qin, Takuya Ueda, Roland Krause, Kaori Yamamoto, Renate Albrecht, Markus Pech, and Knud H. Nierhaus
Until now, two initiation modes for bacterial translation have been described: (i) the standard 30S-binding mode, where the small ribosomal subunit selects the initiation site on an mRNA with the help of three initiation factors (IFs), and (ii) the rare initiation of leaderless mRNAs, which are mRNAs carrying the initiation AUG within the first 5 nt at the 5′-end. The existence of a third “70S-scanning” mode for bacterial initiation was conjectured in past decades but has remained experimentally unproven. Here, we demonstrate the existence of a 70S-scanning mode of initiation and characterize its mechanistic features. The three initiation modes demonstrate specific patterns of requirements for IF1 and IF3. (See pp. E1180–E1189.)
Mechanism of a cytosolic O-glycosyltransferase essential for the synthesis of a bacterial adhesion protein
Yu Chen, Ravin Seepersaud, Barbara A. Bensing, Paul M. Sullam, and Tom A. Rapoport
Protein O-glycosylation is an important process in all cells. Substrates are often modified at multiple Ser/Thr residues, but how a glycosyltransferase can act on a continuously changing substrate is unknown. Here, we have analyzed the mechanism by which the cytosolic O-glycosyltransferase GtfA/B of Streptococcus gordonii modifies the Ser/Thr-rich repeats of adhesin, a protein that mediates the attachment of the bacterium to host cells. GtfA/B is a tetramer, with two molecules of GtfA and GtfB. The GtfB subunit of the glycosyltransferase provides the primary polypeptide-binding site, whereas GtfA performs catalysis. GtfB binds unmodified substrate when conformationally constrained by GtfA and binds modified adhesin molecules when in a relaxed conformation. This model explains how the glycosyltransferase can modify a progressively changing substrate molecule. (See pp. E1190–E1199.)
Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation
Shimin Jiang, Akihiro Narita, David Popp, Umesh Ghoshdastider, Lin Jie Lee, Ramanujam Srinivasan, Mohan K. Balasubramanian, Toshiro Oda, Fujiet Koh, Mårten Larsson, and Robert C. Robinson
Actins and tubulins have dedicated functions that vary between eukaryotes and prokaryotes. During cell division, the prokaryotic contractile ring depends on the tubulin-like protein FtsZ, whereas this task relies on actin in eukaryotes. In contrast, microtubules orchestrate DNA segregation in eukaryotes, yet prokaryotic plasmid segregation often depends on actin-like proteins; this implies that actins and tubulins have somewhat interchangeable properties. Hence, we sought a bacterial filament that more closely resembles microtubules. Here, we report an actin from Bacillus thuringiensis that forms dynamic, antiparallel, two-stranded supercoiled filaments, which pair in the presence of a binding partner to form hollow cylinders. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable properties to the eukaryotic microtubule. (See pp. E1200–E1205.)
Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading
Marija Iljina, Gonzalo A. Garcia, Mathew H. Horrocks, Laura Tosatto, Minee L. Choi, Kristina A. Ganzinger, Andrey Y. Abramov, Sonia Gandhi, Nicholas W. Wood, Nunilo Cremades, Christopher M. Dobson, Tuomas P. J. Knowles, and David Klenerman
Growing experimental evidence suggests that the pathological spreading of alpha-synuclein aggregates in Parkinson’s disease is mediated through a process of templated seeding whereby aggregates catalyze the conversion of soluble protein molecules into their aggregated forms. A molecular-level understanding of this process is still lacking. Here, we determine the concentrations and numbers of aggregates necessary for the effective seeding of alpha-synuclein, thus providing a quantitative framework to understand the conditions when its seeded propagation is favorable. We find that high concentrations of aggregates are needed for seeding yet that aggregates cause cytotoxicity at significantly lower concentrations. This suggests that templated seeding is unlikely to be the main mechanism of spreading in Parkinson’s disease but occurs together with oligomer-induced cellular stress. (See pp. E1206–E1215.)
Conserved properties of individual Ca2+-binding sites in calmodulin
D. Brent Halling, Benjamin J. Liebeskind, Amelia W. Hall, and Richard W. Aldrich
Calmodulin is essential for sensing intracellular Ca2+ in eukaryotic cells. Calmodulin modulates hundreds of effectors, and it has a highly conserved protein sequence. Humans have three identical copies, but a change in either the protein sequence or the protein expression level of any one of the three copies can cause life-threatening disease. We analyzed calmodulin sequences across eukaryotes and compared biophysical properties and structures to show that all of calmodulin’s four Ca2+-binding sites have conserved properties that distinguish them from one another. (See pp. E1216–E1225.)
MEKK2 mediates an alternative β-catenin pathway that promotes bone formation
Matthew Blake Greenblatt, Dong Yeon Shin, Hwanhee Oh, Ki-Young Lee, Bo Zhai, Steven P. Gygi, Sutada Lotinun, Roland Baron, Dou Liu, Bing Su, Laurie H. Glimcher, and Jae-Hyuck Shim
Here, we report a previously unidentified alternative pathway mediated by mitogen-activated protein kinase kinase kinase 2 (MEKK2) for the activation of β-catenin in osteoblasts that is distinct from the classical glycogen synthase kinase 3β (GSK3β)-mediated degradation pathway. The GSK3β pathway regulates β-catenin stability via β-catenin ubiquitination, and its inhibition prevents β-catenin degradation. The MEKK2 pathway instead rescues ubiquitinated β-catenin from degradation via deubiquitination. This pathway plays a critical role in controlling bone mass in vivo, and targeting this pathway may offer an attractive approach for the therapeutic regulation of β-catenin activity. (See pp. E1226–E1235.)
Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect
Hiva Fassihi, Mieran Sethi, Heather Fawcett, Jonathan Wing, Natalie Chandler, Shehla Mohammed, Emma Craythorne, Ana M. S. Morley, Rongxuan Lim, Sally Turner, Tanya Henshaw, Isabel Garrood, Paola Giunti, Tammy Hedderly, Adesoji Abiona, Harsha Naik, Gemma Harrop, David McGibbon, Nicolaas G. J. Jaspers, Elena Botta, Tiziana Nardo, Miria Stefanini, Antony R. Young, Robert P. E. Sarkany, and Alan R. Lehmann
Xeroderma pigmentosum (XP) is a genetic disorder caused by defective repair of DNA damage. Affected patients are mutated in one of eight genes and develop skin pigmentation changes, skin cancers, ocular surface abnormalities, and, in some cases, acute sunburn and neurodegeneration. The XP proteins are involved in different steps in the repair of DNA damage. Examination of 89 patients, the largest reported cohort under long-term follow-up, by the same multidisciplinary team of clinicians and scientists has revealed unexpected clinical heterogeneity dependent on the affected gene and the exact mutation. Our findings provide new insights into the mechanisms of carcinogenesis, ocular surface disease, and neurodegeneration, as well as providing improved clinical management and more definitive prognostic predictions. (See pp. E1236–E1245.)
Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary
Allison K. Timmons, Albert A. Mondragon, Claire E. Schenkel, Alla Yalonetskaya, Jeffrey D. Taylor, Katherine E. Moynihan, Jon Iker Etchegaray, Tracy L. Meehan, and Kimberly McCall
Programmed cell death is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Here we demonstrate that a specific example of large-scale nonapoptotic developmental programmed cell death in the Drosophila ovary occurs by an alternative cell death program where surrounding epithelial cells nonautonomously promote the death of the germ line. We find that genes normally required for engulfment of dying cells act to promote the death of the germ line. Developmental programmed cell death in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous cell death, providing insight on the diversity of cell death mechanisms. (See pp. E1246–E1255.)
Sex-specific regulation of Lgr3 in Drosophila neurons
Geoffrey W. Meissner, Shengzhan D. Luo, Brian G. Dias, Michael J. Texada, and Bruce S. Baker
For individuals to develop sexually dimorphic body parts and behavior, their cells must know their sex. In the fruit fly Drosophila melanogaster, this process is carried out by a series of genes ending with fruitless (fru) and doublesex (dsx). We found that both Fru and Dsx regulate the expression of the leucine-rich repeat G protein-coupled receptor 3 (Lgr3) gene in separate sets of neurons, including neurons important for female sexual behavior. Thus, Lgr3 is important for sexual development and is a point of convergence after the fru/dsx split. (See pp. E1256–E1265.)
Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells
Salah Mansour, Anna S. Tocheva, Chris Cave-Ayland, Moritz M. Machelett, Barbara Sander, Nikolai M. Lissin, Peter E. Molloy, Mark S. Baird, Gunthard Stübs, Nicolas W. J. Schröder, Ralf R. Schumann, Jörg Rademann, Anthony D. Postle, Bent K. Jakobsen, Ben G. Marshall, Rajendra Gosain, Paul T. Elkington, Tim Elliott, Chris-Kriton Skylaris, Jonathan W. Essex, Ivo Tews, and Stephan D. Gadola
T cells autoreactive to cluster of differentiation 1c (CD1c) are abundant in human blood but lipid antigens recognized by these T cells remained poorly understood. A new 2.4-Å structure of CD1c and computational simulations thereof indicated substantial conformational plasticity of CD1c with ligand-induced formation of an F′ roof and G′ portal, as well as the potential of CD1c to present acylated sterols. Confirming these predictions we demonstrated CD1c loading and biophysical interaction of CD1c–lipid complexes with self-reactive human T-cell receptors for two lipid classes: cholesteryl esters similar to those accumulating in foamy macrophages (e.g., in atherosclerosis) and acylated steryl glycosides from Borrelia burgdorferi. These findings differentiate CD1c from other CD1 isoforms and open up new avenues for research into the role of CD1c in human immunity. (See pp. E1266–E1275.)
How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire
Sydney J. Blevins, Brian G. Pierce, Nishant K. Singh, Timothy P. Riley, Yuan Wang, Timothy T. Spear, Michael I. Nishimura, Zhiping Weng, and Brian M. Baker
T-cell receptor (TCR) recognition of antigenic peptides presented by major histocompatibility complex (MHC) proteins defines specificity in cellular immunity. Evidence suggests that TCRs are intrinsically biased toward MHC proteins, yet how this bias coexists alongside the considerable structural variability that is necessary for TCRs to engage different ligands has been a longstanding puzzle. By examining structural and sequence data, we found evidence that human αβ TCRs have an inherent compatibility with structural and chemical properties of MHC proteins. This compatibility leads TCRs to an intrinsic MHC bias but does not compel the formation of particular modes of binding, providing a solution to how TCRs can be MHC-biased but still structurally adaptable. (See pp. E1276–E1285.)
Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets
John F. Ryan, Rachel Hovde, Jacob Glanville, Shu-Chen Lyu, Xuhuai Ji, Sheena Gupta, Robert J. Tibshirani, David C. Jay, Scott D. Boyd, R. Sharon Chinthrajah, Mark M. Davis, Stephen J. Galli, Holden T. Maecker, and Kari C. Nadeau
The mechanisms through which successful immunotherapy induces possible deletion, replacement, or reprogramming of T cells are unknown. By evaluating the expression of T-cell–related genes, and using appropriate multivariate statistical approaches, our data show that successful immunotherapy can induce previously unidentified CD4+ T-cell subtypes during treatment that could help to predict an “immune-tolerant” clinical phenotype identified after cessation of treatment. The ability to use “anergic” transcriptional phenotypes in single T cells to predict successful “immune tolerance” induction in the clinic setting, as suggested by our findings, could lead to transformative impacts in the field of immunotherapy. (See pp. E1286–E1295.)
Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma
Yonathan Lissanu Deribe, Yanxia Shi, Kunal Rai, Luigi Nezi, Samir B. Amin, Chia-Chin Wu, Kadir C. Akdemir, Mozhdeh Mahdavi, Qian Peng, Qing Edward Chang, Kirsti Hornigold, Stefan T. Arold, Heidi C. E. Welch, Levi A. Garraway, and Lynda Chin
Mutations in the PI3K/PTEN/Akt signaling pathway occur frequently across multiple tumor types. These mutations primarily serve to activate PI-3 and Akt kinases. PREX2 is a guanine nucleotide exchanger for Rac1 that is significantly mutated in melanoma and pancreatic ductal adenocarcinoma. Here we report that a mouse model of a truncating PREX2 mutation shows accelerated melanoma development in the context of mutant NRAS. Truncating PREX2 mutations have increased Rac1 guanine nucleotide exchange factor activity, and tumors harboring these mutations have elevated PI3K/Akt pathway activation and reduced expression of critical negative cell cycle regulators leading to increased cell proliferation. This work provides evidence for a previously unidentified mechanism of activating Rac1, the PI3K pathway, and regulation of cell cycle progression in melanoma. (See pp. E1296–E1305.)
Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice
Jun Li, Cecilia Ying Ju Sung, Nikki Lee, Yueqiong Ni, Jussi Pihlajamäki, Gianni Panagiotou, and Hani El-Nezami
Hepatocellular carcinoma is the second most deadly cancer type globally, requiring the development of alternative or complementary therapeutic and prophylactic methods. Here, when feeding a mouse model with a novel probiotic mixture 1 wk before the tumor inoculation, we observed a reduction of the tumor weight and size by 40% compared with the control. Our results revealed that the probiotics’ beneficial effect is closely related with the abundance of certain beneficial bacteria that produce antiinflammatory metabolites, which subsequently regulate the proinflammatory immune cell population via the crosstalk between gut and tumor. We believe that our study highlights the extraordinary potential of probiotics in extraintestine cancers and can be adapted to the study of other cancers. (See pp. E1306–E1315.)
The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function
Samuel E. Marsh, Edsel M. Abud, Anita Lakatos, Alborz Karimzadeh, Stephen T. Yeung, Hayk Davtyan, Gianna M. Fote, Lydia Lau, Jason G. Weinger, Thomas E. Lane, Matthew A. Inlay, Wayne W. Poon, and Mathew Blurton-Jones
Neuroinflammation and activation of innate immunity are pathological hallmarks of Alzheimer’s disease (AD). In contrast, very few studies have examined the impact of the adaptive immune system in AD pathogenesis. Here, we find that genetic ablation of peripheral immune cell populations significantly accelerates amyloid pathogenesis, worsens neuroinflammation, and alters microglial activation state. Critically, it appears that loss of IgG-producing B cells impairs microglial phagocytosis, thereby exacerbating amyloid deposition. Conversely, replacement of IgGs via direct injection or bone marrow transplantation reverses these effects and reduces Aβ pathology. Together, these results highlight the importance of the adaptive immune system and its interactions with microglia in the pathogenesis of AD. (See pp. E1316–E1325.)