Exploring the Potential of Cryo-Electron Tomography on Protein Nanocrystals for Molecular Structure Determination

The three-dimensional structure of a protein molecule, challenging to determine and close to impossible to predict, plays a key role in understanding protein function and has implications in drug design. When it comes to structure determination, there exist many complementary methods, each with their specific advantages and disadvantages. Most of those methods rely on a combined signal from thousands of individuals and cannot be used for directly reconstructing an actual 3d volume as it appears inside the sample. This thesis focuses on developing the methodology and providing proof of concept for a novel approach in structure determination by reconstructing small protein nanocrystals via cryo-electron tomography. Real-space imaging gets past the phase problem that is a challenging companion of conventional diffraction-based methods. With electron tomography we can reconstruct and visualize a 3d nanocrystal in its entirety and study the properties of small biological crystal from a new perspective. Being a relatively unexplored territory, nanocrystal tomography sets several challenges, such as creating nanocrystals small enough for imaging with transmission electron microscope and developing algorithms for going from a tilt-series to a 3d structure. For a proof of concept we create, image and reconstruct nanocrystals of hen egg white lysozyme that, having molecular weight of only 15 kDa, is generally considered unfeasible for electron tomography. Nanocrystals make finding and determining the relative orientations of the individual molecules possible, symmetry relations help reduce the effects of missing information, and by averaging we are able to reconstruct a molecular structure at a medium resolution of around 13 Å. Using Fourier Transform (FFT) we get a direct objective measure of the resolution of details within the reconstruction in the form of a diffraction pattern and show that in specific directions the resolution reaches as high as 7 Å in a single tomogram. Additionally, this work explores two other tightly related ideas. First, we study the concept of extended field and show with extensive simulations that extending the reconstruction space in various regularized iterative reconstruction procedures helps reduce the overall error and prevent over-smoothing. Second, calculating FFT of an image comes at a computational cost, and when the image is not periodic, the discontinuity of the opposing edges causes undesirable strong artifacts in the FFT that could obstruct important details. In this project we implemented a simultaneous 2d FFT and edge artifact removal for real-time applications on a Field Programmable Gate Array (FPGA) reconfigurable computing system.Okinawa Institute of Science and Technology Graduate Universit

Uued võimalused inimese papilloomiviiruse replikatsiooni tõkestavate ühendite kirjeldamiseks

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Peaaegu kõik inimesed nakatuvad oma eluaja jooksul Inimese papilloomiviirusega (HPV), mille erinevaid tüüpe on tänaseks kirjeldatud rohkem kui 200. Suur osa HPV nakkusi möödub kas asümptomaatiliselt või tekivad selle käigus healoomulised vohandid (soolatüükad), mis on küll ebameeldivad, aga mitte eluohtlikud ning üldiselt immuunsüsteemi abil „kaovad“. Soolatüügaste ravimiseks on eestlastel juba ammusest ajast efektiivsed viisid välja töötatud: tuleb neid lihtsalt hõõruda vastu midagi, mida valgustab täiskuu. Siiski võib püsiv HPV nakkus viia aga mitmesuguste pahaloomuliste kasvajate tekkeni; kõige levinumaks on emakakaelavähk, mille tagajärjel hukkub igal aastal sadu tuhandeid naisi. Enamike kõige ohtlikumate HPV tüüpide nakkusi on võimalik ennetada vaktsineerimisega, kuid efektiivne ravi käimasoleva infektsiooni tõkestamiseks puudub. Tänapäeval identifitseeritakse uusi ravimeid läbi keemiliste ainete raamatukogude, mis sisaldavad tuhandeid ühendeid, sõeluuringute. Klassikalised HPV replikatsiooni mõõtmise meetodid selliseid uuringuid teha ei võimalda. Antud töö käigus loodi mudelsüsteem, mis võimaldab hinnata tuhandete keemiliste ühendite mõju HPV replikatsioonile, kasutades lihtsalt mõõdetavaid reportergeene Renilla ja Firefly lutsiferaas. Seda süsteemi kasutades identifitseeriti viis keemilist ühendid, mis tõkestavad spetsiifiliselt vähki tekitavate HPV-de replikatsiooni. Nende ühendite analüüsi tulemusena kirjeldati ka uudsed märklaudvalgud, mis on hõlmatud HPV replikatsioonikompleksi ning mille inhibeerimiseks disainitud ravimid võimaldavad tõkestada viiruse infektsiooni.Most individuals at some point during their life will be infected with Human Papillomaviruses (HPVs). Usually the infection is asymptomatic or results in the formation of benign lesions like warts, which will be cleared by the immune system. However, in rare cases, infection with High-risk HPVs becomes persistent and can eventually cause various carcinomas, most commonly cervical carcinomas. There are more than half a million new cervical carcinoma cases diagnosed every year. HPV infection could be prevented through vaccination but there is no cure against an ongoing infection. Drug development usually begins by screening chemical libraries containing thousands of compounds, against the target of interest. This kind of screening requires specific assay systems. Most important outcome of this work was the development of such a model system for anti-HPV drug screening. This system uses easily quantifiable reporter genes Renilla and Firefly luciferase to describe the effect of the compound on HPV replication. Using this system five new compounds specifically inhibiting the replication of High-risk HPVs. Analyses of the target proteins of those compounds revealed new pathways which are absolutely necessary for HPV replication; and which could be used as targets when designing new anti-HPV compounds

An Extended Field-Based Method for Noise Removal From Electron Tomographic Reconstructions

Molecular structure determination is important for understanding functionalities and dynamics of macromolecules, such as proteins and nucleic acids. Cryo-electron tomography (ET) is a technique that can be used to determine the structures of individual macromolecules, thus providing the snapshots of their native conformations. Such 3-D reconstructions encounter several types of imperfections due to missing, corrupted, and low-contrast data. In this paper, we demonstrate that extending the reconstruction space, which increases the dimensionality of the linear system being solved during reconstruction, facilitates the separation of signal and noise. A considerable amount of the noise associated with collected projection data arises independently from the geometric constraint of image formation, whereas the solution to the reconstruction problem must satisfy such geometric constraints. Increasing the dimensionality thereby allows for a redistribution of such noise within the extended reconstruction space, while the geometrically constrained approximate solution stays in an effectively lower dimensional subspace. Employing various tomographic reconstruction methods with a regularization capability we performed extensive simulation and testing and observed that enhanced dimensionality significantly improves the accuracy of the reconstruction. Our results were validated with reconstructions of colloidal silica nanoparticles as well as P. falciparum erythrocyte membrane protein 1. Although the proposed method is used in the context of Cryo-ET, the method is general and can be extended to a variety of other tomographic modalities

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1a

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located pro-tein with cytoprotective effects in neurons and pancreatic b cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1a, and we identify the binding interface between MANF and IRE1a. The expression of wild-type MANF, but not its IRE1a binding-deficient mutant, attenuates UPR signaling by decreasing IRE1a oligomerization; phosphor-ylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1a interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1a as an intracellular receptor for MANF and regulator of neuronal survival.Peer reviewe

Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic beta-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1-9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive.Peer reviewe

The Cell Cycle Timing of Human Papillomavirus DNA Replication.

Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research

The transcription map of human papillomavirus type 18 during genome replication in U2OS cells.

The human osteosarcoma cell line U2OS is useful for studying genome replication of human papillomavirus (HPVs) subtypes that belong to different phylogenetic genera. In this study, we defined the HPV18 transcription map in U2OS cells during transient replication, stable maintenance and vegetative amplification by identifying viral promoter regions, transcription polyadenylation and splicing sites during HPV18 genome replication. Mapping of the HPV18 transcription start sites in U2OS cells revealed five distinct promoter regions (P102, P520, P811, P1193 and P3000). With the exception of P3000, all of these regions have been previously identified during productive HPV18 infection. Collectively, the data suggest that U2OS cells are suitable for studying the replication and transcription properties of HPVs and to serve as a platform for conducting high-throughput drug screens to identify HPV replication inhibitors. In addition, we have identified mRNA species that are initiated from the promoter region P3000, which can encode two E2C regulator proteins that contain only the C-terminal hinge and DNA-binding and dimerization domains of E2. We show that these proteins regulate the initial amplification of HPV18 by modulating viral transcription. Moreover, we show that one of these proteins can act as a transcriptional activator of promoter P102

Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay.

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers