l-Hydroxyproline (l-Hyp) is a nonstandard amino acid that is present in certain proteins, in some antibiotics and in the cell-wall components of plants. l-Hyp is the product of the post-translational modification of protein prolines by prolyl hydroxylase enzymes, and the isomers trans-3-hydroxy-l-proline (T3LHyp) and trans-4-hydroxy-l-proline (T4LHyp) are major components of mammalian collagen. T4LHyp follows two distinct degradation pathways in bacteria and mammals, while T3LHyp is metabolized by a two-step metabolic pathway that is conserved in bacteria and mammals, which involves a T3LHyp dehydratase and a Δ1-pyrroline-2-carboxylate (Pyr2C) reductase. In order to shed light on the structure and catalysis of the enzyme involved in the second step of the T3LHyp degradation pathway, the crystal structure of Pyr2C reductase from the archaeon Thermococcus litoralis DSM 5473 complexed with NADH and l-proline is presented. The model allows the mapping of the residues involved in cofactor and product binding and represents a valid model for rationalizing the catalysis of Pyr2C reductases.

Aldo-keto reductases (AKRs) are NADPH/NADP+-dependent oxidoreductase enzymes that metabolize an aldehyde/ketone to the corresponding alcohol. AKR4C14 from rice exhibits a much higher efficiency in metabolizing malondialdehyde (MDA) than do the Arabidopsis enzymes AKR4C8 and AKR4C9, despite sharing greater than 60% amino-acid sequence identity. This study confirms the role of rice AKR4C14 in the detoxification of methylglyoxal and MDA, and demonstrates that the endogenous contents of both aldehydes in transgenic Arabidopsis ectopically expressing AKR4C14 are significantly lower than their levels in the wild type. The apo structure of indica rice AKR4C14 was also determined in the absence of the cofactor, revealing the stabilized open conformation. This is the first crystal structure in AKR subfamily 4C from rice to be observed in the apo form (without bound NADP+). The refined AKR4C14 structure reveals a stabilized open conformation of loop B, suggesting the initial phase prior to cofactor binding. Based on the X-ray crystal structure, the substrate- and cofactor-binding pockets of AKR4C14 are formed by loops A, B, C and β1α1. Moreover, the residues Ser211 and Asn220 on loop B are proposed as the hinge residues that are responsible for conformational alteration while the cofactor binds. The open conformation of loop B is proposed to involve Phe216 pointing out from the cofactor-binding site and the opening of the safety belt. Structural comparison with other AKRs in subfamily 4C emphasizes the role of the substrate-channel wall, consisting of Trp24, Trp115, Tyr206, Phe216, Leu291 and Phe295, in substrate discrimination. In particular, Leu291 could contribute greatly to substrate selectivity, explaining the preference of AKR4C14 for its straight-chain aldehyde substrate.

The biological processes related to protein homeostasis in Mycobacterium tuberculosis, the etiologic agent of tuberculosis, have recently been established as critical pathways for therapeutic intervention. Proteins of particular interest are ClpC1 and the ClpC1–ClpP1–ClpP2 proteasome complex. The structure of the potent antituberculosis macrocyclic depsipeptide ecumicin complexed with the N-terminal domain of ClpC1 (ClpC1-NTD) is presented here. Crystals of the ClpC1-NTD–ecumicin complex were monoclinic (unit-cell parameters a = 80.0, b = 130.0, c = 112.0 Å, β = 90.07°; space group P21; 12 complexes per asymmetric unit) and diffracted to 2.5 Å resolution. The structure was solved by molecular replacement using the self-rotation function to resolve space-group ambiguities. The new structure of the ecumicin complex showed a unique 1:2 (target:ligand) stoichiometry exploiting the intramolecular dyad in the α-helical fold of the target N-terminal domain. The structure of the ecumicin complex unveiled extensive interactions in the uniquely extended N-terminus, a critical binding site for the known cyclopeptide complexes. This structure, in comparison with the previously reported rufomycin I complex, revealed unique features that could be relevant for understanding the mechanism of action of these potential antituberculosis drug leads. Comparison of the ecumicin complex and the ClpC1-NTD-L92S/L96P double-mutant structure with the available structures of rufomycin I and cyclomarin A complexes revealed a range of conformational changes available to this small N-terminal helical domain and the minor helical alterations involved in the antibiotic-resistance mechanism. The different modes of binding and structural alterations could be related to distinct modes of action.

Death-associated protein kinase 1 (DAPK1) is a serine/threonine protein kinase that regulates apoptosis and autophagy. DAPK1 is considered to be a therapeutic target for amyloid-β deposition, endometrial adenocarcinomas and acute ischemic stroke. Here, the potent inhibitory activity of the natural anthraquinone purpurin against DAPK1 phosphorylation is shown. Thermodynamic analysis revealed that while the binding affinity of purpurin is similar to that of CPR005231, which is a DAPK1 inhibitor with an imidazopyridazine moiety, the binding of purpurin was more enthalpically favorable. In addition, the inhibition potencies were correlated with the enthalpic changes but not with the binding affinities. Crystallographic analysis of the DAPK1–purpurin complex revealed that the formation of a hydrogen-bond network is likely to contribute to the favorable enthalpic changes and that stabilization of the glycine-rich loop may cause less favorable entropic changes. The present findings indicate that purpurin may be a good lead compound for the discovery of inhibitors of DAPK1, and the observation of enthalpic changes could provide important clues for drug development.

As command module pilot for the Apollo 15 mission to the moon in 1971, Al Worden spent six days orbiting the moon, including three days completely alone, the most isolated human in existence. In Falling to Earth, Worden tells for the first time the full story around the dramatic events that shook NASA and ended his spaceflight career.

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Yinlong Song, Yikan Zhang, Ying Pan, Jianfeng He, Yan Wang, Wei Chen, Jing Guo, Haiteng Deng, Yi Xue, Xianyang Fang, and Xin Liang

Microtubules dynamics is regulated by the plus end-tracking proteins (+TIPs) in cells. End binding protein 1 (EB1) acts as a master regulator in +TIPs networks by targeting microtubule growing ends and recruiting other factors. However, the molecular mechanism of how EB1 binds to microtubule ends with a high affinity remains to be an open question. Using single-molecule imaging, we show that the end-binding kinetics of EB1 changes along with the polymerizing and hydrolysis rate of tubulin dimers, confirming the binding of EB1 to GTP/GDP-Pi tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than monomeric EB1 mutants, suggesting that two CH domains in the dimer contribute to the end-binding. Introducing phosphomimicking mutations into the linker domain of EB1 weakens the end-binding affinity and confers a more curved conformation to EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for the end-binding affinity. Taken together, our results provide insights into understanding how the high-affinity end-binding of EB1 can be achieved and how this activity may be regulated in cells.

Imke K. Mandemaker, Di Zhou, Serena T. Bruens, Dick H. Dekkers, Pernette J. Verschure, Raghu R. Edupuganti, Eran Meshorer, Jeroen A. Demmers, and Jurgen A. Marteijn

Many chromatin remodeling and modifying proteins are involved in the DNA damage response by stimulating repair or inducing DNA damage signaling. Interestingly, here we identified that down regulation of the H1-interacting protein SET results in increased resistance to a wide variety of DNA damaging agents. We found that this increased resistance is not the result of an inhibitory effect of SET on DNA repair, but rather the consequence of a suppressed apoptotic response to DNA damage. We further provide evidence that the histone chaperone SET is responsible for the eviction of H1 from chromatin. Knock down of H1 in SET-depleted cells resulted in re-sensitization of cells to DNA damage, suggesting that the increased DNA damage resistance in SET-depleted cells is the result of enhanced retention of H1 on chromatin. Finally, clonogenic survival assays show that SET and p53 are epistatic in attenuating DNA damage-induced cell death. Altogether, our data show a role for SET in the DNA damage response as a regulator of cell survival following genotoxic stress.

Olivia R. Grafinger, Genya Gorshtein, Tyler Stirling, Megan I. Brasher, and Marc G. Coppolino

Malignant cancer cells can invade extracellular matrix (ECM) through the formation of F-actin-rich subcellular structures termed invadopodia. ECM degradation at invadopodia is mediated by matrix metalloproteinases (MMPs), and recent findings indicate that membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a primary role in this process. Maintenance of an invasive phenotype is dependent on internalization of MT1-MMP from the plasma membrane and its recycling to sites of ECM remodeling. Internalization of MT1-MMP is dependent on its phosphorylation, and here we examine the role of β1 integrin-mediated signaling in this process. Activation of β1 integrin using the antibody P4G11 induced phosphorylation and internalization of MT1-MMP and resulted in increased cellular invasiveness and invadopodium formation in vitro. We also observed phosphorylation of Src and epidermal growth factor receptor (EGFR) and an increase in their association in response to β1 integrin activation, and determined that Src and EGFR promote phosphorylation of MT1-MMP on Thr⁵⁶⁷. These results suggest that MT1-MMP phosphorylation is regulated by a β1 integrin-Src-EGFR signaling pathway that promotes recycling of MT1-MMP to sites of invadopodia formation during cancer cell invasion.

Benjamin Ziman, Peter Karabinis, Paul Barghouth, and Nestor J. Oviedo

Nutrient availability upon feeding leads to an increase in body size in the planarian Schmidtea mediterranea. However, it remains unclear how food consumption integrates with cell division at the organismal level. Here we show that Sirtuins is evolutionarily conserved in planarians and specifically demonstrate that Sirtuin-1 (Smed-Sirt-1) regulates organismal growth by impairing both feeding behavior and intestinal morphology. Disruption of Smed-Sirt-1 with either RNAi or pharmacological treatment leads to reduced animal growth. Conversely, enhancement of Smed-Sirt-1 with resveratrol accelerates growth. Differences in growth rates were associated with changes in the amount of time to locate food and overall consumption. Furthermore, Smed-Sirt-1(RNAi) animals displayed reduced cell death and increased stem cell proliferation accompanied by impaired expression of intestinal lineage progenitors and reduced branching of the gut. Altogether, our findings indicate Sirtuin-1 is a crucial metabolic hub capable of controlling animal behavior, tissue renewal and morphogenesis of the adult intestine.

Christian Renz, Veronique Albanese, Vera Tröster, Thomas K. Albert, Olivier Santt, Susan C. Jacobs, Anton Khmelinskii, Sebastien Leon, and Helle D. Ulrich

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only ubiquitin protein ligase (E3) known to cooperate with Ubc13-Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. We now report a contribution of Ubc13-Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13-Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3s for Ubc13-Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13-Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13-Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting.

Kristina Drizyte-Miller, Jing Chen, Hong Cao, Micah B. Schott, and Mark A. McNiven

Epithelial cells such as liver-resident hepatocytes rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rabs can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports mTORC1 signaling under basal and amino acid stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling.

Hem Sapkota, Jonathan D. Wren, and Gary J. Gorbsky

Centrosomes focus microtubules to promote mitotic spindle bipolarity, a critical requirement for balanced chromosome segregation. Comprehensive understanding of centrosome function and regulation requires a complete inventory of components. While many centrosome components have been identified, others may yet remain undiscovered. We have used a bioinformatics approach, based on "guilt by association" expression to identify novel mitotic components among the large group of predicted human proteins that have yet to be functionally characterized. Here we identify Chondrosarcoma-Associated Gene 1 (CSAG1) in maintaining centrosome integrity during mitosis. Depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles more effectively in cells with compromised p53 function. Thus, CSAG1 may reflect a class of "mitotic addiction" genes whose expression is more essential in transformed cells.

Ana Loncar, Sergio A. Rincon, Manuel Lera Ramirez, Anne Paoletti, and Phong T. Tran

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases like cancer or developmental disorders. Here we compared mitotic and meiotic spindles in fission yeast. We show that even though mitotic and meiotic spindles undergo the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of kinesin-14 Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of kinesin-5 Cut7 remains constant. We identified the second kinesin-14 Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines differences between mitotic and meiotic spindles in fission yeast, and provides prospect for future comparative studies.

M. Pinar and M. A. Penalva

TRAnsport Protein Particle (TRAPP) complexes regulate membrane traffic. TRAPPII and TRAPPIII share a core hetero-heptamer, also denoted TRAPPI. In fungi TRAPPIII and TRAPPII mediate GDP exchange on RAB1 and RAB11, respectively, regulating traffic across the Golgi, with TRAPPIII also activating RAB1 in autophagosomes. Our finding that Aspergillus nidulans TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI/TRAPPIII already residing in the Golgi matures into TRAPPII to determine a RAB1-to-RAB11 conversion as Golgi cisternae progress from early Golgi to TGN identity. By time-resolved microscopy we determine that the TRAPPII reporter Trs120/TRAPPC9 is recruited to existing TGN cisternae slightly before RAB11 arrives, and resides for~45 sec on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3/TRAPPC3 was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120/TRAPPC9, indicating en bloc recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.

Kelly L. Dunlevy, Valentina Medvedeva, Jade E. Wilson, Mohammed Hoque, Trinity Pellegrin, Adam Maynard, Madison M. Kremp, Jason S. Wasserman, Andrey Poleshko, and Richard A. Katz

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via "tethering proteins" that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation-dephosphorylation of the LBD. Furthermore, we identified a PP2A phosphatase recognition motif within the evolutionarily conserved PRR14 C-terminal Tantalus domain. Disruption of this motif affected PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable, modular domains. The findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.

Samantha-Su Z. Taylor, Nicole L. Jacobsen, Tasha K. Pontifex, Paul Langlais, and Janis M. Burt

Connexin 37 (Cx37) expression profoundly suppresses proliferation of rat insulinoma (Rin) cells in a manner dependent on gap junction channel (GJCh) functionality and the presence and phosphorylation status of its carboxyl-terminus (CT). In Rin cells growth arrested by induced Cx37 expression, serine 319 (S319) is frequently phosphorylated. Preventing phosphorylation at this site (alanine substitution; S319A) relieved Cx37 of its growth suppressive effect whereas mimicking phosphorylation at this site (aspartate substitution; S319D) enhanced Cx37's growth suppressive properties. Like Cx37-WT, -S319D GJChs and hemichannels (HChs) preferred the closed state, rarely opening fully, and gated slowly. In contrast, Cx37-S319A channels preferred open states, opened fully, and gated rapidly. These data indicate that phosphorylation-dependent conformational differences in Cx37 protein and channel function underlie Cx37-induced growth arrest vs. growth permissive phenotypes. That the closed state of -WT and Cx37-S319D GJChs and HChs favors growth arrest suggests that rather than specific permeants mediating cell cycle arrest, the closed conformation instead supports interaction of Cx37 with growth regulatory proteins that result in growth arrest.

Ganlu Deng, Yihong Chen, Cao Guo, Ling Yin, Ying Han, Yiyi Li, Yaojie Fu, Changjing Cai, Hong Shen, and Shan Zeng

Epithelial-mesenchymal transition (EMT) is a crucial process for cancer cells to acquire metastatic potential, which primarily causes death in gastric cancer (GC) patients. Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family that plays an indispensable role in human cancers. However, little is known about its roles in GC metastasis. In this study, BMP4 was found to be frequently overexpressed in GC tissues and was correlated with patient's poor prognosis. BMP4 was upregulated in GC cell lines and promoted EMT and metastasis of GC cells both in vitro and in vivo, while knockdown of BMP4 significantly inhibited EMT and metastasis of GC cells. Meanwhile, the inhibitor of DNA binding 1 (Id1) was identified as a downstream target of BMP4 by PCR arrays and upregulated via Smad1/5/8 phosphorylation. Id1 knockdown attenuated BMP4-induced EMT and invasion in GC cells. Moreover, Id1 overexpression in BMP4 knockdown cells restored the promotion of EMT and cell invasion. In summary, BMP4 induced EMT to promote GC metastasis by upregulating Id1 expression. Antagonizing BMP4 may be a potential therapeutic strategy in GC metastasis.

Smithsonian Institution Libraries has recently acquired a rare first edition of Darwin's Geological Observations on the Volcanic Islands, Visited During the Voyage of the H.M.S. Beagle.

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The Smithsonian’s National Museum of Natural History recently acquired four remarkable gemstones and jewelry pieces for the Smithsonian’s National Gem Collection in celebration of the 100th anniversary of the museum.

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The Smithsonian Research Online program recently surpassed the mark of 10,000 publications in the Digital Repository. This collection of digital publications by Smithsonian staff represents a broad review of research done by researchers at the Institution.

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Left-handed snails, giant wombats, spiny trilobites, zombie ants, glyptodonts…these are a few of the fascinating animals and plants whose fossils spring to life across the […]

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A 3-D rendering of the Apollo 11 Command Module with a view inside the hatch. The seats inside are where astronauts Neil Armstrong, Buzz Aldrin […]

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The post Many years of research are celebrated in the December 2010 birth of two cheetah cubs at the Smithsonian Conservation Biology Institute appeared first on Smithsonian Insider.

The post After a bulldozer unearthed five statues in Ain Ghazal, Jordan in 1984, Smithsonian conservators carefully restore these otherworldy figures appeared first on Smithsonian Insider.

The post Smithsonian geologist Liz Cottrell discusses the March 11, 2011 Japan earthquake and its mechanics appeared first on Smithsonian Insider.

Paleoecologist Conrad Labandeira travels to the Karoo Basin of South Africa to find leaf fossils from the Permian-Triassic boundary, the time of the Earth's largest mass extinction. What can bug bites on leaves tell us about our own uncertain times?

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The post Killer carnivores: Titanoboa vs. T-Rex — Premieres April 1 on Smithsonian Channel appeared first on Smithsonian Insider.

The post Smithsonian anthropologist JoAllyn Archambault speaks about the outstanding research of 19th-century anthropologist James Mooney appeared first on Smithsonian Insider.

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William Fitzhugh - Edward Nelson's 1877-1881 Western Alaskan Expedition

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The post Japanese acrobats ca. 1927, footage from the Smithsonian’s Human Studies Film Archive appeared first on Smithsonian Insider.

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This film is from the collections of the Human Studies Film Archives, Smithsonian Institution. Clip from silent film, Beautiful Japan (1917-18), by travel-lecturer Benjamin Brodsky. Benten Festival is celebrated on Shiraishi Island. Benten (Benzaiten) is the Goddess of the Sea and one of the Seven Lucky Gods of Japan.

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Silent black & white archival film clip from "Japan: Promotional and Theatrical Footage, ca. 1927". The full film, which is 17 minutes long, includes segments from what are believed to be a theatrical film, a promotional film and, possibly, amateur film - all of unknown origin.

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The post Kepler 11: A Six-Planet Sonata by Alex Parker, postdoctoral researcher at the Harvard–Smithsonian Center for Astrophysics appeared first on Smithsonian Insider.

One of Italy’s greatest treasures, Leonardo da Vinci’s Codex on the Flight of Birds, will be exhibited at the National Air and Space Museum from […]

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Click photo to take a tour of the 1903 Wright Flyer with Smithsonian X 3D Explorer. The Smithsonian X 3D Collection features objects from the […]

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The National Air and Space Museum’s 2014 Trophy Award Winner for Current Achievement goes to the Dawn Flight Team. The Dawn team has successfully performed […]

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The National Air and Space Museum will reopen the “Boeing Milestones of Flight Hall” July 1 in conjunction with the museum’s 40th anniversary. The two-year […]

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“The Outwin 2016: American Portraiture Today” exhibition is on view at the Smithsonian’s National Portrait Gallery from March 12, 2016 – January 8, 2017. The Outwin […]

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To mark the 47th anniversary of the Apollo 11 moon-landing mission, the Smithsonian has made available a high-resolution 3-D scan of the command module “Columbia,” […]

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Jennifer Trask engages nature as both medium and subject matter, combining unexpected materials such as bone, vertebrae, butterfly wings, resin, metal, and antique frame fragments […]

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