Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1
Shih-Hon Li, Ashley A. Reinke, Karen L. Sanders, Cory D. Emal, James C. Whisstock, Jeanne A. Stuckey, and Daniel A. Lawrence
Serine protease inhibitors (serpins) are a protein superfamily whose members are involved in many diseases and are thus attractive drug targets. In addition to protease inhibition, serpins also bind a variety a of other biological molecules, including extracellular matrix components and cell surface receptors. The inhibitory mechanism of serpins requires a conformational change that can also alter their affinity to non-protease ligands. Here (pp. E4941–E4949) a surprising allosteric mechanism of action is revealed for a small molecule inhibitor of the serpin, plasminogen activator inhibitor 1 (PAI-1). Compound binding prevents PAI-1 interaction with both proteases and with its cofactor even though the binding sites are located 40 Å apart. These results suggest the potential for the identification other therapeutically useful serpin inhibitors.
Nfatc1 orchestrates aging in hair follicle stem cells
Brice E. Keyes, Jeremy P. Segal, Evan Heller, Wen-Hui Lien, Chiung-Ying Chang, Xingyi Guo, Dan S. Oristian, Deyou Zheng, and Elaine Fuchs
Signs of aging often first appear in our skin and hair. As animals age, hair follicles spend more time resting instead of generating hair. Here (pp. E4950–E4959) we show that this decline is rooted in age-related changes in systemic, local, and intrinsic factors, which collaborate to reduce hair follicle stem cell (HFSC) activity. We uncover a unique and hitherto-undescribed age-related role for bone morphogenic protein signaling and a downstream effector, nuclear factor of activated T-cell c1 (NFATc1). In young stem cells, NFATc1 is on when they are quiescent and wanes when they make hair. In aging follicles, NFATc1 and its target genes remain high too long. Importantly, NFATc1 inhibitors restore youthful behavior to aging HFSCs, providing unique insights into age-related changes in skin physiology.
Sensitivity to ocean acidification parallels natural pCO2 gradients experienced by Arctic copepods under winter sea ice
Ceri N. Lewis, Kristina A. Brown, Laura A. Edwards, Glenn Cooper, and Helen S. Findlay
The Arctic Ocean is a bellwether for ocean acidification, yet few direct Arctic studies have been carried out and limited observations exist, especially in winter. We present unique under-ice physicochemical data showing the persistence of a mid water column area of high CO2 and low pH through late winter, Zooplankton data demonstrating that the dominant copepod species are distributed across these different physicochemical conditions, and empirical data demonstrating that these copepods show sensitivity to pCO2 that parallels the range of natural pCO2 they experience through their daily vertical migration behavior. Our data (pp. E4960–E4967), collected as part of the Catlin Arctic Survey, provide unique insight into the link between environmental variability, behavior, and an organism’s physiological tolerance to CO2 in key Arctic biota.
RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia
Tao Zu, Yuanjing Liu, Monica Bañez-Coronel, Tammy Reid, Olga Pletnikova, Jada Lewis, Timothy M. Miller, Matthew B. Harms, Annet E. Falchook, S. H. Subramony, Lyle W. Ostrow, Jeffrey D. Rothstein, Juan C. Troncoso, and Laura P. W. Ranum
A GGGGCC expansion mutation located in intron 1 of chromosome 9 ORF 72 (C9ORF72) was recently described as a common cause of familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We show (pp. E4968–E4977) that this single mutation results in the accumulation of sense and antisense RNA foci plus six expansion proteins expressed by repeat-associated non-ATG (RAN) translation. RNAs accumulate in nuclear foci and the RAN proteins form cytoplasmic aggregates in neurons that often cluster in affected brain regions. These results indicate that bidirectional transcription and RAN translation are fundamental pathologic features of C9ORF72 ALS/FTD. Additionally these data have broad implications that change our understanding of how microsatellite expansion mutations are expressed in patient cells and how they cause disease.
Ascites analysis by a microfluidic chip allows tumor-cell profiling
Vanessa M. Peterson, Cesar M. Castro, Jaehoon Chung, Nathan C. Miller, Adeeti V. Ullal, Maria D. Castano, Richard T. Penson, Hakho Lee, Michael J. Birrer, and Ralph Weissleder
Serial molecular analyses of tumor cells during treatment- and biopsy-driven clinical trials are emerging norms for many cancers. Yet surgical and image-guided biopsies are expensive and invasive, explaining why alternative sources for tumor cells are being sought. In ovarian cancer (and other abdominopelvic cancers), abdominal fluid buildup (ascites) occurs frequently. We demonstrate (pp. E4978–E4986) that ascites tumor cells (ATCs) present a valuable source of tumor cells, rendering ascites another form of “liquid biopsy.” We evaluated 85 ovarian cancer-related markers and developed a unique, low cost miniaturized microfluidic ATC chip for on-chip enrichment and molecular profiling using small amounts of ascites. This approach could expand the utility of ATCs within cytotoxic and/or molecularly targeted ovarian cancer therapeutic trials.
The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time
Lina Josefsson, Susanne von Stockenstrom, Nuno R. Faria, Elizabeth Sinclair, Peter Bacchetti, Maudi Killian, Lorrie Epling, Alice Tan, Terence Ho, Philippe Lemey, Wei Shao, Peter W. Hunt, Ma Somsouk, Will Wylie, Daniel C. Douek, Lisa Loeb, Jeff Custer, Rebecca Hoh, Lauren Poole, Steven G. Deeks, Frederick Hecht, and Sarah Palmer
Identifying the source and dynamics of persistent HIV-1 during combinational antiretroviral therapy (cART) is crucial for understanding the barriers to curing HIV infection. Through genetic characterization of HIV-1 DNA in infected cells from peripheral blood and gut-associated lymphoid tissue from patients after long-term suppressive cART, our study (pp. E4987–E4996) reveals that the primary barrier to a cure is a remarkably stable pool of infected memory CD4+ T cells. Through in-depth phylogenetic analyses, we determined that the HIV-1 reservoir in these cells from eight patients is kept stable during long-term cART and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.
Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine
Daniel C. Freed, Qi Tang, Aimin Tang, Fengsheng Li, Xi He, Zhao Huang, Weixu Meng, Lin Xia, Adam C. Finnefrock, Eberhard Durr, Amy S. Espeseth, Danilo R. Casimiro, Ningyan Zhang, John W. Shiver, Dai Wang, Zhiqiang An, and Tong-Ming Fu
Congenital human cytomegalovirus (HCMV) infection is an important cause of newborn disability, and developing a vaccine against congenital HCMV is a top priority. However, despite decades of efforts, a vaccine remains elusive. Previous vaccines lacked an antigen called pentameric glycoprotein H (gH) complex, essential for the virus to infect epithelial/endothelial cells, and these vaccines induced poor neutralizing antibodies. To support a unique vaccine concept featuring the pentameric gH complex, we established 45 mAbs from a rabbit immunized with an experimental vaccine. Over 50% of the mAbs have antiviral activity, and potent clones target the pentameric gH complex, thus establishing this antigen as the key for potent antiviral antibodies by vaccination. Our result (pp. E4997–E5005) contributes to the understanding of immune attributes of an effective vaccine against HCMV.
Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection
Sarah L. Grady, John G. Purdy, Joshua D. Rabinowitz, and Thomas Shenk
Virus infection can trigger profound alterations in host cell metabolism, but the role of individual enzymes in this process is relatively unstudied. Here, we show that argininosuccinate synthetase 1 (AS1) antagonizes the production of herpes simplex virus 1 (HSV-1) in cultured fibroblasts. Infection reduces the level of AS1 protein, and further reduction induced by siRNA treatment enhances the production of HSV-1. AS1 deficiency mimics many of the metabolic changes induced by HSV-1 infection, demonstrating that a decrease in the activity of a single cellular enzyme is responsible for much of the metabolic reprogramming induced by HSV-1. These results (pp. E5006–E5015) underscore the dependence of HSV-1 replication on altered host cell metabolism.
Crystallographic insights into sodium-channel modulation by the β4 subunit
John Gilchrist, Samir Das, Filip Van Petegem, and Frank Bosmans
Voltage-gated sodium (Nav) channels are members of a large complex that plays a crucial role in rapid electrical signaling throughout the human body. As prominent members of this complex, β-subunits modify Nav channel function and cause debilitating disorders when mutated. Collectively, the functional and crystallographic results reported in this work uncover intricate interactions of these elements within the Nav-channel signaling complex and establish a key role for β-subunits in shaping Nav1.2 pharmacology. An important concept emerging from our results is (pp. E5016–E5024) that β-subunits provide exciting opportunities for designing new therapeutic strategies to correct their abnormal behaviors.
DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis
Daniela S. Floss, Julien G. Levy, Véronique Lévesque-Tremblay, Nathan Pumplin, and Maria J. Harrison
Arbuscular mycorrhizal (AM) symbiosis is a mutualistic interaction formed between most land plants and soil fungi. During symbiosis the fungus develops branched hyphae, known as arbuscules, inside the root cortical cells. Arbuscules are critical to the symbiosis and function in phosphate delivery to the plant. Here we show that arbuscule formation is regulated by DELLA proteins. DELLA proteins are negative regulators of gibberellic acid (GA) signaling and repress plant growth and development. Our data (pp. E5025–E5034) provide insights into regulation of arbuscule formation and identify a potential mechanism by which the plant can coordinate the symbiosis with its growth and nutrient status.