Browsing by Subject "STRUCTURAL BASIS"

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  • Fewer, David P.; Metsä-Ketelä, Mikko (2020)
    Abstract Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.
  • Vincenzi, Bruno; Napolitano, Andrea; Fiocco, Marta; Mir, Olivier; Rutkowski, Piotr; Blay, Jean-Yves; Reichardt, Peter; Joensuu, Heikki; Fumagalli, Elena; Gennatas, Spyridon; Hindi, Nadia; Nannini, Margherita; Ceruso, Mariella Spalato; Italiano, Antoine; Grignani, Giovanni; Brunello, Antonella; Gasperoni, Silvia; De Pas, Tommaso; Badalamenti, Giuseppe; Pantaleo, Maria A.; van Houdt, Winan J.; IJzerman, Nikki S.; Steeghs, Neeltje; Gelderblom, Hans; Desar, Ingrid M. E.; Falkenhorst, Johanna; Silletta, Marianna; Sbaraglia, Marta; Tonini, Giuseppe; Martin-Broto, Javier; Hohenberger, Peter; Le Cesne, Axel; Jones, Robin L.; Dei Tos, Angelo P.; Gronchi, Alessandro; Bauer, Sebastian; Casali, Paolo G. (2022)
    Purpose: The effect of high-dose imatinib (800 mg/day) on survival in the adjuvant treatment of patients with resected KIT exon 9-mutated gastrointestinal stromal tumors (GIST) is not established. Here, the association of dose and other clinicopatho-logic variables with survival was evaluated in a large multi-institutional European cohort. Experimental Design: Data from 185 patients were retrospec-tively collected in 23 European GIST reference centers. Propen-sity score matching (PSM) and inverse-probability of treatment weighting (IPTW) were used to account for confounders. Uni-variate and multivariate unweighted and weighted Cox propor-tional hazard regression models were estimated for relapse-free survival (RFS), modified-RFS (mRFS) and imatinib failure-free survival (IFFS). Univariate Cox models were estimated for overall survival. Results: Of the 185 patients, 131 (70.8%) received a starting dose of 400 mg/d and the remaining 54 (29.2%) a dose of 800 mg/d. Baseline characteristics were partially unbalanced, suggesting a potential selection bias. PSM and IPTW analyses showed no advantage of imatinib 800 mg/d. In the weighted multivariate Cox models, high-dose imatinib was not associated with the survival outcomes [RFS: hazard ratio (HR), 1.24; 95% confidence interval (CI), 0.79-1.94; mRFS: HR, 1.69; 95% CI, 0.92-3.10; IFFS: HR, 1.35; 95% CI, 0.79- 2.28]. The variables consistently associated with worse survival out-comes were high mitotic index and nongastric tumor location. Conclusions: In this retrospective series of patients with KIT exon 9-mutated GIST treated with adjuvant imatinib, a daily dose of 800 mg versus 400 mg did not show better results in terms of survival outcomes. Prospective evaluation of the more appropriate adjuvant treatment in this setting is warranted.
  • Pawlowski, Rafal; Rajakylä, Eeva; Vartiainen, Maria K.; Treisman, Richard (2010)
  • Sahu, Biswajyoti; Pihlajamaa, Paivi; Dubois, Vanessa; Kerkhofs, Stefanie; Claessens, Frank; Jänne, Olli A. (2014)
  • Pantsar, Tatu; Rissanen, Sami; Dauch, Daniel; Laitinen, Tuomo; Vattulainen, Ilpo; Poso, Antti (2018)
    A mutated KRAS protein is frequently observed in human cancers. Traditionally, the oncogenic properties of KRAS missense mutants at position 12 (G12X) have been considered as equal. Here, by assessing the probabilities of occurrence of all KRAS G12X mutations and KRAS dynamics we show that this assumption does not hold true. Instead, our findings revealed an outstanding mutational bias. We conducted a thorough mutational analysis of KRAS G12X mutations and assessed to what extent the observed mutation frequencies follow a random distribution. Unique tissue-specific frequencies are displayed with specific mutations, especially with G12R, which cannot be explained by random probabilities. To clarify the underlying causes for the nonrandom probabilities, we conducted extensive atomistic molecular dynamics simulations (170 its) to study the differences of G12X mutations on a molecular level. The simulations revealed an allosteric hydrophobic signaling network in KRAS, and that protein dynamics is altered among the G12X mutants and as such differs from the wild-type and is mutation-specific. The shift in long-timescale conformational dynamics was confirmed with Markov state modeling. A G12X mutation was found to modify KRAS dynamics in an allosteric way, which is especially manifested in the switch regions that are responsible for the effector protein binding. The findings provide a basis to understand better the oncogenic properties of KRAS G12X mutants and the consequences of the observed nonrandom frequencies of specific G12X mutations.
  • Hattula, Katarina; Hirschberg, Daniel; Kalkkinen, Nisse; Butcher, Sarah J.; Ora, Ari (2014)
  • Euro, Liliya; Haapanen, Outi; Rog, Tomasz; Vattulainen, Ilpo; Suomalainen, Anu; Sharma, Vivek (2017)
    DNA polymerase gamma (Pol gamma) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol gamma replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory beta-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol gamma-associated neurodegenerative disorders.
  • Martin, Isabel M.; Nava, Michele M.; Wickström, Sara A.; Graeter, Frauke (2022)
    Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component integrin-linked kinase (ILK) dynamically responds to mechanical force and what role adenosine triphosphate (ATP) bound to this pseudokinase plays remain elusive. Here, we apply force-probe molecular-dynamics simulations of human ILK:alpha-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key salt-bridge-forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.
  • Qiu, Tianyi; Yang, Yiyan; Qiu, Jingxuan; Huang, Yang; Xu, Tianlei; Xiao, Han; Wu, Dingfeng; Zhang, Qingchen; Zhou, Chen; Zhang, Xiaoyan; Tang, Kailin; Xu, Jianqing; Cao, Zhiwei (2018)
    Major challenges in vaccine development include rapidly selecting or designing immunogens for raising cross-protective immunity against different intra-or inter-subtypic pathogens, especially for the newly emerging varieties. Here we propose a computational method, Conformational Epitope (CE)-BLAST, for calculating the antigenic similarity among different pathogens with stable and high performance, which is independent of the prior binding-assay information, unlike the currently available models that heavily rely on the historical experimental data. Tool validation incorporates influenza-related experimental data sufficient for stability and reliability determination. Application to dengue-related data demonstrates high harmonization between the computed clusters and the experimental serological data, undetectable by classical grouping. CE-BLAST identifies the potential cross-reactive epitope between the recent zika pathogen and the dengue virus, precisely corroborated by experimental data. The high performance of the pathogens without the experimental binding data suggests the potential utility of CE-BLAST to rapidly design cross-protective vaccines or promptly determine the efficacy of the currently marketed vaccine against emerging pathogens, which are the critical factors for containing emerging disease outbreaks.
  • Raivola, Juuli; Haikarainen, Teemu; Silvennoinen, Olli (2020)
    The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-gamma (IFN gamma) and interferon-alpha (IFN alpha) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFN gamma signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the alpha C-helix. Mutating L633 at the alpha C reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.
  • Seifert, Tina; Malo, Marcus; Kokkola, Tarja; Engen, Karin; Friden-Saxin, Maria; Wallen, Erik A. A.; Lahtela-Kakkonen, Maija; Jarho, Elina M.; Luthman, Kristina (2014)
  • Sah-Teli, Shiv K.; Hynönen, Mikko J.; Schmitz, Werner; Geraets, James A.; Seitsonen, Jani; Pedersen, Jan Skov; Butcher, Sarah J.; Wierenga, Rik K.; Venkatesan, Rajaram (2019)
    The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid beta-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.
  • Karhu, Lasse; Magarkar, Aniket; Bunker, Alex; Xhaard, Henri (2019)
    We assess the stability of two previously suggested binding modes for the neuropeptide orexin-A in the OX2 receptor through extensive molecular dynamics simulations. As the activation determinants of the receptor remain unknown, we simulated an unliganded receptor and two small-molecular ligands, the antagonist suvorexant and the agonist Nag26 for comparison. Each system was simulated in pure POPC membrane as well as in the 25% cholesterol–POPC membrane. In total, we carried out 36 μs of simulations. Through this set of simulations, we report a stable binding mode for the C-terminus of orexin-A. In addition, we suggest interactions that would promote orexin receptor activation, as well as others that would stabilize the inactive state.
  • Sanz, Dafne Jacome; Raivola, Juuli; Karvonen, Hanna; Arjama, Mariliina; Barker, Harlan; Murumägi, Astrid; Ungureanu, Daniela (2021)
    Simple Summary Ovarian cancer (OC) is known for its poor prognosis, due to the absence of reliable biomarkers and its late diagnosis, since the early-stage disease is almost asymptomatic. Lipid metabolism plays an important role in OC progression due to the development of omental metastasis in the abdominal cavity. The aim of our study was to assess the therapeutic role of various enzymes involved in lipid metabolism regulation or synthesis, in different subtypes of OC represented by cell lines as well as patient-derived cancer cell cultures (PDCs). We show that proprotein convertase subtilisin/kexin type 9 (PCSK9), a cholesterol-regulating enzyme, plays a pro-survival role in OC and targeting its expression impairs cancer cell growth. We also tested a small library of metabolic and mTOR-targeting drugs to identify drug vulnerabilities specific to various subtypes of OC. Our results show that in OC cell lines and PDCs the second generation of mTOR inhibitors such as AZD8055, vistusertib, dactolisib and sapanisertib, have higher cytotoxic activity compared to the first generation mTOR inhibitors such as rapalogs. These results suggest that, in the era of precision medicine, it is possible to target the metabolic pathway in OC and identify subtype-specific drug vulnerabilities that could be advanced to the clinic. Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic (n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures (PDCs, n = 5) to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements.
  • Javanainen, Matti; Martinez-Seara, Hector; Vattulainen, Ilpo (2017)
    The coarse-grained Martini model is employed extensively to study membrane protein oligomerization. While this approach is exceptionally promising given its computational efficiency, it is alarming that a significant fraction of these studies demonstrate unrealistic protein clusters, whose formation is essentially an irreversible process. This suggests that the protein-protein interactions are exaggerated in the Martini model. If this held true, then it would limit the applicability of Martini to study multi-protein complexes, as the rapidly clustering proteins would not be able to properly sample the correct dimerization conformations. In this work we first demonstrate the excessive protein aggregation by comparing the dimerization free energies of helical transmembrane peptides obtained with the Martini model to those determined from FRET experiments. Second, we show that the predictions provided by the Martini model for the structures of transmembrane domain dimers are in poor agreement with the corresponding structures resolved using NMR. Next, we demonstrate that the first issue can be overcome by slightly scaling down the Martini protein-protein interactions in a manner, which does not interfere with the other Martini interaction parameters. By preventing excessive, irreversible, and non-selective aggregation of membrane proteins, this approach renders the consideration of lateral dynamics and protein-lipid interactions in crowded membranes by the Martini model more realistic. However, this adjusted model does not lead to an improvement in the predicted dimer structures. This implicates that the poor agreement between the Martini model and NMR structures cannot be cured by simply uniformly reducing the interactions between all protein beads. Instead, a careful amino-acid specific adjustment of the protein-protein interactions is likely required.
  • Mobarak, Edouard; Håversen, Liliana; Manna, Moutusi; Rutberg, Mikael; Levin, Malin; Perkins, Rosie; Rog, Tomasz; Vattulainen, Ilpo; Boren, Jan (2018)
    Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol-and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.
  • van Belkum, Alex; Almeida, Carina; Bardiaux, Benjamin; Barrass, Sarah V.; Butcher, Sarah J.; Çaykara, Tuğçe; Chowdhury, Sounak; Datar, Rucha; Eastwood, Ian; Goldman, Adrian; Goyal, Manisha; Izadi-Pruneyre, Nadia; Jacobsen, Theis; Johnson, Pirjo H.; Kempf, Volkhard A.J.; Kiessling, Andreas; Bueno, Juan Leva; Malik, Anchal; Malmström, Johan; Meuskens, Ina; Milner, Paul A.; Nilges, Michael; Pamme, Nicole; Peyman, Sally A.; Rodrigues, Ligia R.; Rodriguez-Mateos, Pablo; Sande, Maria G.; Silva, Carla Joana; Stehle, Thilo; Thibau, Arno; Vaca, Diana J.; Linke, Dirk (2021)
    Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.
  • Anastasina, Maria; Le May, Nicolas; Bugai, Andrii; Fu, Yu; Soderholm, Sandra; Gaelings, Lana; Ohman, Tiina; Tynell, Janne; Kyttanen, Suvi; Barboric, Matjaz; Nyman, Tuula A.; Matikainen, Sampsa; Julkunen, Ilkka; Butcher, Sarah J.; Egly, Jean-Marc; Kainov, Denis E. (2016)
    Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication. (C) 2016 Elsevier B.V. All rights reserved.