Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps
Pratyush Tiwary, Vittorio Limongelli, Matteo Salvalaglio, and Michele Parrinello
A crucial factor for drug efficacy is not just the binding affinity, but also the mean residence time in the binding pocket, usually quantified by its inverse, koff. This is an important parameter that regulates the time during which the drug is active. Whereas the calculation of the binding affinity is by now routine, the calculation of koff has proven more challenging because the timescales involved far exceed the limits of standard molecular dynamics simulation. We propose (pp. E386–E391) a metadynamics-based strategy that allows reaching timescales of seconds, and estimate koff along with unbinding pathways and associated dynamical bottlenecks. The protocol is exemplified for trypsin–benzamidine unbinding. This work is a step towards a more effective computer-based drug design.
Rapid deposition of oxidized biogenic compounds to a temperate forest
Tran B. Nguyen, John D. Crounse, Alex P. Teng, Jason M. St. Clair, Fabien Paulot, Glenn M. Wolfe, and Paul O. Wennberg
Dry deposition is an important removal mechanism for oxidized atmospheric compounds. This process remains, however, poorly understood due to the scarcity of direct flux observations for all but small, inorganic molecules in the atmosphere. The chemically speciated fluxes presented here (pp. E392–E401) comprise a unique and novel dataset that quantifies the dry deposition velocities for a variety of trace gases in a typical forested ecosystem. The data illustrate the key role of molecular diffusion in the atmosphere−biosphere exchange of water-soluble species. Furthermore, this work enabled evaluation of the dry deposition parameterization in a global chemical transport model. The results aid in resolving key discrepancies within the global model, resulting in more-accurate predictions of trace gas lifetimes and surface concentrations.
Jagged–Delta asymmetry in Notch signaling can give rise to a Sender/Receiver hybrid phenotype
Marcelo Boareto, Mohit Kumar Jolly, Mingyang Lu, José N. Onuchic, Cecilia Clementi, and Eshel Ben-Jacob
Notch signaling pathway plays crucial roles in cell-fate determination during embryonic development and cancer progression. According to the current paradigm, the Notch–Delta signaling leads to complementary cell-fate selection between two neighboring cells where one acts as Sender or Receiver. However, this picture is not complete because an additional ligand, Jagged, is involved in the Notch signaling. We devise (pp. E402–E409) a specific theoretical framework to decipher the functional role of Jagged. We find that the asymmetry between the modulations of Delta and Jagged leads to the existence of the previously unexplored possibility of a Sender–Receiver phenotype enabling two interacting cells to share a similar fate. This realization can provide important clues regarding embryonic development, wound healing, and how to target tumor–stroma signaling.
Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions
Celine Walmacq, Lanfeng Wang, Jenny Chong, Kathleen Scibelli, Lucyna Lubkowska, Averell Gnatt, Philip J. Brooks, Dong Wang, and Mikhail Kashlev
Cyclopurines are bulky oxidative DNA lesions that strongly block RNA polymerase II (Pol II) to interfere with gene transcription and replication in mammalian cells. Cells developed a mechanism enabling slow transcriptional bypass of the cyclopurines. Similar to translesion synthesis by DNA polymerases, lesion bypass by Pol II can be highly mutagenic, leading to transcription errors that are reminiscent of the A rule. We elucidated (pp. E410–E419) the mechanism and determined the domain in Pol II responsible for error-free and error-prone lesion bypass. We also identified a positive role of mammalian factor TFIIF in lesion-bypass stimulation. Strikingly, Pol II uses a similar strategy for negotiation with different DNA lesions, such as cyclopurines, pyrimidine dimers, cisplatin, and abasic sites, to reduce the burden of DNA damage on genome stability.
MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells
Diego Ploper, Vincent F. Taelman, Lidia Robert, Brian S. Perez, Björn Titz, Hsiao-Wang Chen, Thomas G. Graeber, Erika von Euw, Antoni Ribas, and Edward M. De Robertis
MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a driver of lysosomal biogenesis, we found (pp. E420–E429) that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation.
Spata6 is required for normal assembly of the sperm connecting piece and tight head–tail conjunction
Shuiqiao Yuan, Clifford J. Stratton, Jianqiang Bao, Huili Zheng, Bhupal P. Bhetwal, Ryuzo Yanagimachi, and Wei Yan
Male infertility due to acephalic spermatozoa has been reported in both animals and humans, but its cause remains largely unknown. Here (pp. E430–E439) we report that inactivation of Spata6, an evolutionarily conserved gene, in mice leads to failure in development of the connecting piece during late spermiogenesis, along with production of headless spermatozoa in the epididymis and ejaculates. The defects may be ascribed to the disrupted myosin-based microfilament transport mediated by SPATA6 through its interactions with myosin light-chain and heavy-chain subunits. This study not only unveils the process of sperm neck formation at both the ultrastructural and molecular levels, but also provides a genetic basis for the production of acephalic spermatozoa in both humans and animals.
Real-time resolution of point mutations that cause phenovariance in mice
Tao Wang, Xiaowei Zhan, Chun-Hui Bu, Stephen Lyon, David Pratt, Sara Hildebrand, Jin Huk Choi, Zhao Zhang, Ming Zeng, Kuan-wen Wang, Emre Turer, Zhe Chen, Duanwu Zhang, Tao Yue, Ying Wang, Hexin Shi, Jianhui Wang, Lei Sun, Jeff SoRelle, William McAlpine, Noelle Hutchins, Xiaoming Zhan, Maggy Fina, Rochelle Gobert, Jiexia Quan, McKensie Kreutzer, Stephanie Arnett, Kimberly Hawkins, Ashley Leach, Christopher Tate, Chad Daniel, Carlos Reyna, Lauren Prince, Sheila Davis, Joel Purrington, Rick Bearden, Jennifer Weatherly, Danielle White, Jamie Russell, Qihua Sun, Miao Tang, Xiaohong Li, Lindsay Scott, Eva Marie Y. Moresco, Gerald M. McInerney, Gunilla B. Karlsson Hedestam, Yang Xie, and Bruce Beutler
In forward genetics, a mutagen is used to randomly induce germline mutations that cause variant phenotypes. Forward genetics permits discovery of genes necessary for biological phenomena, but identifying the mutations that cause variant phenotypes is time-consuming and in the past usually occurred long after the phenotype was first recognized. Here (pp. E440–E449) we introduce a method and software tool, Linkage Analyzer, for identifying causative mutations present in the germline of mutant mice concurrent with recognition of variant phenotypes. It requires knowledge of genotype at all mutation sites in members of a pedigree prior to phenotypic assessment. Using this method and software, forward genetic studies in mice are limited only by the rates of mutant production and screening.
Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell
Rashmi Kumar, Martina P. Bach, Federica Mainoldi, Mikako Maruya, Satoshi Kishigami, Hassan Jumaa, Teruhiko Wakayama, Osami Kanagawa, Sidonia Fagarasan, and Stefano Casola
Antibodies produced by B cells provide a protective barrier to our organism against the penetration and dissemination of microorganisms. Each antibody recognizes a specific antigen through variable (V) region domains of pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In mammals, VDJ recombination generates a highly diversified preimmune pool of VH and VL domains. Acquisition of a functional VH rearrangement is thought to prevent further VDJ recombination at the IgH locus. Instead, mice cloned from a terminally differentiated B cell unravel the ability of VDJ recombination to revise a functionally rearranged VH gene through VH replacement. We show (pp. E450–E457) that up to 20% of the antibody V gene repertoire of mature B-lymphocytes can be generated through VH replacement.
VH replacement in primary immunoglobulin repertoire diversification
Amy Sun, Tatiana I. Novobrantseva, Maryaline Coffre, Susannah L. Hewitt, Kari Jensen, Jane A. Skok, Klaus Rajewsky, and Sergei B. Koralov
The recombinatorial process of V(D)J rearrangement generates a vast antibody repertoire from a limited number of genes. The joints generated in the course of V(D)J recombination are imprecise thus yielding greater diversity but also resulting in frequent generation of nonproductive VDJ rearrangements. We have previously shown that B cells with two nonproductive IgH rearrangements can be efficiently rescued by a form of secondary V(D)J recombination called VH replacement. We now demonstrate (pp. E458–E466) that VH replacement also contributes to the diversity of the immune repertoire by modifying productive IgH rearrangements. Results presented herein suggest that VH replacement occurs exclusively during early stages of B-cell development and therefore does not contribute to the editing of self-reactive antibodies.
Fear and C-reactive protein cosynergize annual pulse increases in healthy adults
Shani Shenhar-Tsarfaty, Nadav Yayon, Nir Waiskopf, Itzhak Shapira, Sharon Toker, David Zaltser, Shlomo Berliner, Ya'acov Ritov, and Hermona Soreq
Increased heart rate predicts all-cause mortality; however, the major causes for elevated basal heart rate values and annual changes in them have not been systematically studied, possibly because of the high individual variability involved. Our study (pp. E467–E471) addressed the question, “which physiological and psychological parameters determine basal pulse and its annual changes?,” in a cohort of 17,380 apparently healthy volunteers. This is, to the best of our knowledge, the first statistics-based search for the major interactions between physiological and psychological determinants of basal pulse and annual pulse changes; it indicates, perhaps not surprisingly, that consistent exposure to terror threats ignites fear-induced exacerbation of preexisting neuro-immune risks of all-cause mortality and proposes a set of risk-predicting parameters that may have translational value.
Combating pertussis resurgence: One booster vaccination schedule does not fit all
Maria A. Riolo and Pejman Rohani
Pertussis has reemerged as a major public health concern in many countries where vaccine uptake remains high and pertussis has been considered well controlled until recently. In our paper (pp. E472–E477), we address the important scientific and practical problem of developing optimal booster vaccination schedules by using a genetic algorithm. Our results argue that booster vaccination schedules developed based on misdiagnosis of the problem are likely to be epidemiologically ineffective and economically costly.
Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone
Xiaolin Tu, Jesus Delgado-Calle, Keith W. Condon, Marta Maycas, Huajia Zhang, Nadia Carlesso, Makoto M. Taketo, David B. Burr, Lilian I. Plotkin, and Teresita Bellido
Bone anabolic stimuli activate canonical Wnt/β-catenin signaling and human mutations of this pathway underscore its essential role in bone accrual. However, the cell responsible for orchestrating these actions has remained elusive. This study (pp. E478–E486) identifies osteocytes as mediators of the anabolic effect of canonical Wnt/β-catenin signaling in bone; further, it dissects desired (bone gain) from undesired (decreased resorption and leukemia) outcomes by selective activation of canonical Wnt/β-catenin signaling in osteocytes versus osteoblasts. Long-lived osteocytes constitute more than 90% of bone cells, thus representing more logical and effective target cells to induce bone anabolism than scarce, short-lived osteoblasts. These findings pave the way toward the development of better treatments for osteoporosis and other bone-related disorders targeting osteocytic Wnt/β-catenin signaling.
Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials
Guillaume Cornelis, Cécile Vernochet, Quentin Carradec, Sylvie Souquere, Baptiste Mulot, François Catzeflis, Maria A. Nilsson, Brandon R. Menzies, Marilyn B. Renfree, Gérard Pierron, Ulrich Zeller, Odile Heidmann, Anne Dupressoir, and Thierry Heidmann
Syncytins are “captured” genes of retroviral origin, corresponding to the fusogenic envelope gene of endogenized retroviruses. They are present in a series of eutherian mammals, including humans and mice where they play an essential role in placentation. Here (pp. E487–E496) we show that marsupials—which diverged from eutherian mammals ∼190 Mya but still possess a primitive, short-lived placenta (rapidly left by the embryo for development in an external pouch)—have also captured such genes. The present characterization of the syncytin-Opo1 gene in the opossum placenta, together with the identification of two additional endogenous retroviral envelope gene captures, allow a recapitulation of the natural history of these unusual genes and definitely extends their “symbiotic niche” to all clades of placental mammals.
Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells
Elisa Duregotti, Samuele Negro, Michele Scorzeto, Irene Zornetta, Bryan C. Dickinson, Christopher J. Chang, Cesare Montecucco, and Michela Rigoni
The neuromuscular junction is the site of transmission of the nerve impulse to the muscle. This finely tuned synapse relies on at least three components: the motor neuron, the muscle fiber, and the Schwann cells, which assist nerve recovery after injury. Using animal neurotoxins to induce an acute and reversible nerve degeneration, we have identified (pp. E497–E505) several mitochondrial molecules through which the damaged nerve terminal communicates with nearby cells, activating signaling pathways in Schwann cells involved in nerve regeneration. Among these messengers, hydrogen peroxide appears to be crucial at the initial stages of regeneration, because its inactivation delays the functional recovery of the damaged neuromuscular junction in vivo. These findings provide important indications about the pharmacological treatment of traumatized patients.