The RecQ family of helicases has been termed the “Caretakers of the Genome,” and rightfully so. These proteins are highly conserved from bacteria to humans and have been implicated in functions from homologous recombinatorial repair to damage checkpoint response to telomere maintenance and more. Mutant genes of three of the human RecQ helicases lead to syndromes characterized by a high incidence of cancer, premature aging and early death. Despite their implications in several biological functions and importance to the integrity of the human genome and suppression of cancer, many aspects of the RecQ family structure and function remain unknown. To date, much is known about the catalytic function of the helicase domain and accompanying domains, but considerably less is known about the non-catalytic N-terminus in these proteins, which, in many cases, including those human orthologs involved in disease, can make up about half of the total protein length. While experiments have been able to identify protein partners that interact with the N-terminal region, few are able to narrow the binding sites to minimally functional parts and fewer still describe any detail regarding the structural features of these binding areas. In fact, some reviews have generally described the N-terminus as “featureless,” a concept we challenge in our studies. Many of the N-termini of these RecQs have long been known to contain large stretches of acidic residues, a feature of intrinsically disordered regions. These regions/proteins are rich in charged and polar residues, lack compactness that makes crystallography possible, and have flexible and dynamic conformations that are prevalent in “high specificity, low affinity” interactions. Disordered proteins are well-known to be hot spots for protein/protein interactions and post-translational modifications, amongst other functions. Considering these facts, and recognizing the ties between these and what we know about the N-termini of the RecQs, we hypothesized that these proteins likely have long disordered termini. In Chapter 3, we confirm the presence of disorder at the Top3/Rmi1 binding site on Sgs1, the Saccharomyces cerevisiae RecQ helicase. We show that even in a disordered state, this binding region is not “featureless,” but in fact contains a transient alpha-helical molecular recognition element that is necessary to facilitate complex formation between Sgs1, Top3 and Rmi1. Loss of helical structure at this site leads to increased genomic instability and sensitivity to DNA damaging agents. Based on these results, we suggest that there are likely many more such elements in the N-terminus that that are important for other Sgs1 protein/protein interactions and provide an estimate for the number of interactions in this region. In Chapter 4, we evaluate the prevalence of disorder in a set of Chromatin Processes proteins in an effort to establish a role for disorder with regards to maintaining chromatin integrity. In our bioinformatics study, we found that disorder is overrepresented in the Chromatin Processes proteins, and that a major driving force for disorder in these proteins is protein/protein interaction and post-translational modification. We also show a biological connection to disorder and increased protein/protein interaction by investigating these parameters in the context of the DNA damage checkpoint response and in complex formations. Mediators between highly structured kinases in the checkpoint were the most interactive proteins and over half of all predicted interaction sites occurred in disordered areas. Complexed proteins often contained one protein with a high number of disordered sites and a high number of predicted interactions, while the rest were considerably more ordered. Chapter 5 explores a Sgs1 interaction partner, Rmi1 and uses bioinformatics to design structurally-based point mutations in an effort to further elucidate Rmi1 function in yeast, which remains largely unknown outside of its enhancement of Top3/Sgs1 catalytic function. Using AGADIR, which predicts alpha-helical structure and is particularly useful in our hands for guided-mutagenesis in disordered regions, we identified several point mutations that lead to Δrmi1 phenotypes or intermediate growth on hydroxyurea. We hypothesize that these mutants are important in maintaining Rmi1 stability. Together, these studies suggest an important change in how the field approaches further studies into the RecQ helicases; traditional methods of primary sequence comparisons and crystal structures limit the study of disordered regions that are still functionally important. Future care should be given to consider the conservation of structure or structural elements in the RecQs over strict alignments when comparing functional regions between orthologs. Our studies also suggest that it is highly likely that structural motifs for important protein interactions in RecQs are being overlooked because they are not readily obvious using traditional methods. By understanding these motifs and the interactions they facilitate, we may be able to more easily identify polymorphisms in patients with genomically unstable conditions like cancer and, having better understood the biological process these structures facilitate, design drugs to counteract detrimental effects.

No description available.

In this thesis we implement an ensemble of sequence analysis strategies aimed at identifying functional and structural protein features. The first part of this work was dedicated to two case studies of specific proteins analyzed to provide candidate functional positions for experimental validation: the protein alpha-synuclein (αsyn) and the alanine racemases protein family. In the case of αsyn, the objective was to predict its aggregation prone regions. For the alanine racemase protein family, the scope was to predict sites responsible for substrate specificity. In these two studies, computational predictions allowed systematically exploring potentially functionally relevant protein sites in an efficient manner that may not be possible to implement with traditional experimental approaches. Our strategy provided a powerful forecasting tool for the selection of candidate sites to be later verified experimentally. In the second part, we analyze the role of intrinsic disorder (ID) as a modulator of protein function in different organisms and cellular processes, which is largely unexplored. As key components of the diverse cellular pathways, disordered proteins are often involved in many diseases, including cancer and neurodegenerative diseases. Thus, there is an impeding need to unveil the general principles underlying the role of ID in proteins. We provide a multi-scale analysis of the involvement of ID in protein function starting with a large-scale analysis at genomic level of the role of ID in Arabidopsis, zooming in into the specific processes of vesicular trafficking in Human and yeast, and finally focusing on specific proteins of diverse organisms. The results of this thesis provide a better understanding of the functional roles mediated by ID in different organisms and biological processes, such as acting as flexible linkers connecting structured domains, mediating protein-protein interactions, and assisting the quick assembly of large macromolecular complexes. In addition, we present evidence of the use of ID as a mechanism to increase the complexity of protein and biological networks, and as a means to increase the adaptability of proteins in specific processes. Thus, our results contribute to elucidating the relationship between network and organismal complexity and ID, while they also provide evidence of the evolutionary advantages offered by ID.

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal was to identify NS2 residues important for interaction with the host ubiquitin system, as well as describe the mechanism by which NS2 induces host protein ubiquitination. Bioinformatics analysis provided a platform for development of loss-of-ubiquitin-function NS2 mutants. Combining critical mutations as double or triple NS2 ubiquitin mutants displayed an additive effect on reducing NS2-induced ubiquitination. Recombinant RSV (rRSV) containing NS2 ubiquitin mutations maintained their effect on ubiquitin expression during infection in addition to limiting STAT2 degradation activity. NS2 ubiquitin mutants decreased rRSV growth and increased levels of innate immune responses, indicating a correlation between NS2’s ubiquitin function and antagonism of type I IFN to enhance viral replication. Finally, several proteomics strategies were employed to identify specific cellular proteins ubiquitinated by NS2 to further define host-pathogen interactions during RSV infection. This study demonstrates an effective approach for limiting viral protein function to enhance immune responses during infection.

Malaria is the cause of over one million deaths per year, primarily in African children. The parasite responsible for the most virulent form of malaria, is Plasmodium falciparum. Protein structure plays a pivotal role in elucidating mechanisms of parasite functioning and resistance to anti-malarial drugs. Protein structure furthermore aids the determination of protein function, which can together with the structure be used to identify novel drug targets in the parasite. However, various structural features in P. falciparum proteins complicate the experimental determination of protein three dimensional structures. Furthermore, the presence of parasite-specific inserts results in reduced similarity of these proteins to orthologous proteins with experimentally determined structures. The lack of solved structures in the malaria parasite, together with limited similarities to proteins in the Protein Data Bank, necessitate genome-scale structural annotation of P. falciparum proteins. Additionally, the annotation of a range of structural features facilitates the identification of suitable targets for structural studies. An integrated structural annotation system was constructed and applied to all the predicted proteins in P. falciparum, Plasmodium vivax and Plasmodium yoelii. Similarity searches against the PDB, Pfam, Superfamily, PROSITE and PRINTS were included. In addition, the following predictions were made for the P. falciparum proteins: secondary structure, transmembrane helices, protein disorder, low complexity, coiled-coils and small molecule interactions. P. falciparum protein-protein interactions and proteins exported to the RBC were annotated from literature. Finally, a selection of proteins were threaded through a library of SCOP folds. All the results are stored in a relational PostgreSQL database and can be viewed through a web interface (http://deepthought.bi.up.ac.za:8080/Annotation). In order to select groups of proteins which fulfill certain criteria with regard to structural and functional features, a query tool was constructed. Using this tool, criteria regarding the presence or absence of all the predicted features can be specified. Analysis of the results obtained revealed that P. falciparum protein-interacting proteins contain a higher percentage of predicted disordered residues than non-interacting proteins. Proteins interacting with 10 or more proteins have a disordered content concentrated in the range of 60-100%, while the disorder distribution for proteins having only one interacting partner, was more evenly spread. Comparisons of structural and sequence features between the three species, revealed that P. falciparum proteins tend to be longer and vary more in length than the other two species. P. falciparum proteins also contained more predicted low complexity and disorder content than proteins from P. yoelii and P. vivax. P. falciparumprotein targets for experimental structure determination, comparative modeling and in silico docking studies were putatively identified based on structural features. For experimental structure determination, 178 targets were identi_ed. These targets contain limited contents of predicted transmembrane helix, disorder, coiled-coils, low complexity and signal peptide, as these features may complicate steps in the experimental structure determination procedure. In addition, the targets display low similarity to proteins in the PDB. Comparisons of the targets to proteins with crystal structures, revealed that the structures and predicted targets had similar sequence properties and predicted structural features. A group of 373 proteins which displayed high levels of similarity to proteins in the PDB, were identified as targets for comparitive modeling studies. Finally, 197 targets for in silico docking were identified based on predicted small molecule interactions and the availability of a 3D structure. / Dissertation (MSc)--University of Pretoria, 2008. / Biochemistry / unrestricted

Submitted to the faculty of the Bioinformatics Graduate Program in partial fulfillment of the requirements for the degree Master of Science in the School of Informatics, Indiana University December 2005 / The last decade has witnessed numerous proteomic studies which have predicted and successfully confirmed the existence of extended structurally flexible regions in protein molecules. Parallel to these advancements, the last five years of structural bioinformatics has also experienced an explosion of results on molecular recognition and its importance in protein-protein interactions. This work provides an extension to past and ongoing research efforts by looking specifically at the “flexibility and disorder†found in protein sequences involved in molecular recognition processes and known as, Molecular Recognition Elements or Molecular Recognition Features (MoREs or MoRFs, as we call them). MoRFs are relatively short in length (10 – 70 residues length); loosely structured protein regions within longer sequences that are largely disordered in nature. Interestingly, upon binding to other proteins, these MoRFs are able to undergo disorder-to-order transition. Thus, in our interpretation, MoRFs could serve as potential binding sites, and that this binding to another protein lends a functional advantage to the whole protein complex by enabling interaction with their physiological partner. There are at least three basic types of MoRFs: those that form α-helical structures upon binding, those that form β-strands (in which the peptide forms a β-sheet with additional β-strands provided by the protein partner), and those that form irregular structures when bound. Our proposed names for these structures are α-MoRF (also known as α-MoRE, alpha helical molecular recognition feature/element), β-MoRF (beta sheet molecular recognition feature/element), and I-MoRF (Irregular molecular recognition feature/element), respectively. The results presented in this work suggest that functionally significant residual structure can exist in MoRF regions prior to the actual binding event. We also demonstrate profound conformational preferences within MoRF regions for α-helices. We believe that the results from this study would subsequently improve our understanding of protein-protein interactions especially those related to the molecular recognition, and may pave way for future work on the development of protein binding site predictions. We hope that via the conclusions of this work, we would have demonstrated that within only a few of years of its conception, intrinsic protein disorder has gained wide-scale importance in the field of protein-protein interactions and can be strongly associated with molecular recognition.

[ES] La activación de las diferentes cascadas de señalización en respuesta al estrés ambiental, así como mantener activas las proteínas y complejos proteicos en respuesta al estrés celular es fundamental para las plantas. La chaperona Hsp90 juega un papel importante en la coordinación de estos dos procesos, aunque los mecanismos que regulan su actividad en respuesta al ambiente no están completamente descritos. Estudios recientes en animales muestran que las proteínas prefoldin-like (PFDLs), co-chaperonas de Hsp90, desempeñan un papel importante en la señalización ambiental. Por lo tanto, son capaces de transmitir información sobre el medio ambiente para modular tanto el ensamblaje de complejos proteicos como parte del Hsp90-R2TP/PFDL como las vías de señalización en las que se encuentran involucradas. Hoy en día, se conoce muy poco sobre las proteínas PFDLs en especies vegetales. En este trabajo, hemos obtenido evidencia de que las PFDLs, particularmente URI1, pueden ejercer un papel similar en Arabidopsis, coordinando la homeostasis de las proteínas con las vías de crecimiento en respuesta a diferentes tipos de estrés, como por ejemplo el estrés por falta de energía. Así, mostramos que el complejo R2TP/PFDL se forma en Arabidopsis y que URI1 es una de sus subunidades. La actividad de URI1 es esencial para ciertos procesos, como el desarrollo embrionario, evidenciado por el arresto embrionario temprano causado por la mutación knock-out de URI1. Se ha observado que URI1 tiene una influencia notoria en el transcriptoma mediante el uso de un alelo hipomórfico de uri1. Coherentemente con lo observado en el transcriptoma, el interactoma de URI1 muestra que URI1 interactúa con un número relativamente grande de proteínas, muchas de las cuales están involucradas en procesos fundamentales relacionados con el metabolismo del ARN mensajero y la transducción de señales. URI1 es una proteína altamente versátil, aunque la base molecular de esta versatilidad aún es desconocida. Aquí mostramos que URI1 en Arabidopsis posee una región intrínsicamente desordenada que abarca la mayoría de la parte C-terminal de la proteína, característica que se conserva en los ortólodos de levadura y humanos. Nuestros resultados revelan en URI1 dos características principales de las proteínas desordenadas. La primera de ellas es la promiscuidad en las interacciones con otras proteínas y la segunda la inestabilidad de la proteína. Hipotetizamos que estas dos contribuyen a dotar a URI1 de versatilidad funcional. Es importante destacar que la inestabilidad de URI1 se contrarresta con el azúcar. El análisis genético realizado sitúa a URI1 en la vía de señalización que controla el crecimiento en respuesta al estrés energético inducido por la privación de azúcar, al desempeñar un papel como un regulador negativo aguas arriba de una de las quinasas principales, TOR. Hipotetizamos que URI1 desempeña un papel en la prevención del crecimiento excesivo de las plántulas cuando las condiciones energéticas son favorables. / [CA] És de vital importància per a una planta activar les corresponents cascades de senyalització en resposta a l'estrés ambiental i mantenir actives les proteïnes i els complexos proteics malgrat l'estrés cel·lular. La xaperona Hsp90, entre d'altres, s'encarrega de coordinar aquests dos processos, encara que els mecanismes que regulen la seua activitat en resposta a l'entorn no s'han arribat a comprendre del tot. Estudis recents en animals mostren que les co-xaperones de Hsp90, prefoldin-like (PFDLs), tenen un rol destacat en la senyalització ambiental. Per tant, tenen el potencial de portar informació sobre l'entorn per modular tant l'assemblatge de complexos proteics, com a part de l'Hsp90-R2TP/PFDL, i les vies de senyalització en les quals estan involucrats. Actualment, hi ha poca informació sobre PFDLs en espècies vegetals. Ara tenim l'evidència que els PFDLs, particularment URI1, poden exercir un paper similar en Arabidopsis, coordinant l'homeòstasi proteica amb les vies de creixement en resposta a l'estrés, per exemple, estrés energètic. En aquest treball mostrem que el complex R2TP/PFDL es forma a Arabidopsis i que URI1 és una de les seues subunitats. L'activitat d'URI1 és essencial per a alguns processos, com el desenvolupament embrionari, com es demostra per l'arrest precoç causat per la mutació knock-out d'URI1. Amb un al·lel hipomòrfic d'uri1, es va mostrar que URI1 té una forta influència en el transcriptoma. En consonancia amb l'observat amb el transcriptoma, l'interactoma d'URI1 mostra que URI1 interactua amb un nombre relativament gran de proteïnes, moltes de les quals estan involucrades en processos fonamentals relacionats amb el metabolisme de l'ARN missatger. Així, URI1 en Arabidopsis, com els seus ortòlegs en rent i humans, sembla estar involucrat en diverses funcions cel·lulars, incloent-hi l'homeòstasi proteica, el metabolisme del ARNm i la transducció de senyals. URI1 és una proteïna altament versàtil, encara que la base molecular d'aquesta versatilitat encara és desconeguda. Ací mostrem que Arabidopsis URI1 posseeix una gran regió intrínsecament desordenada que abasta la major part de la porció C-terminal de la proteïna, una característica que es conserva en els ortòlegs de rent i humans. Els nostres resultats revelen dues característiques principals de les proteïnes desordenades en URI1. La primera la promiscuïtat en les interaccions amb altres proteïnes, i la segona la inestabilitat proteica. Aleshores, hipotetitzem que aquestes dues característiques contribueixen a dotar URI1 de versatilitat funcional. Curiosament, la inestabilitat d'URI1 es contraresta amb el sucre, i la nostra anàlisi genètica situa URI1 en la via de senyalització que controla el creixement en resposta a l'estrés energètic induït per la privació de sucre, actuant com a regulador negatiu aigües amunt de la quinasa TOR. Hipotetitzem que URI1 exerceix un rol en prevenir el creixement excessiu de les plàntules quan les condicions energètiques són favorables. / [EN] It is of fundamental importance for the plant to trigger the corresponding signaling cascades in response to environmental stress and to keep proteins and protein complexes active despite the cellular stress. The chaperone Hsp90 plays an important role in coordinating these two processes, although the mechanisms that regulate its activity in response to the environment are not fully understood. Recent studies in animals show that the Hsp90 co-chaperones prefoldin-like (PFDLs) play a role in environmental signaling. Therefore, they have the potential to carry information about the environment to modulate both the assembly of protein complexes as part of the Hsp90-R2TP/PFDL and the signaling pathways in which they are involved. Currently, there is little information on PFDLs in plant species. We have now accumulated evidence that PFDLs, particularly URI1, may exert a similar, general role in Arabidopsis, coordinating protein homeostasis with growth pathways in response to stress, e.g. low energy stress. Here we show that the R2TP/PFDL complex is formed in Arabidopsis and that URI1 is one of its subunits. The activity of URI1 is essential for certain processes, such as embryonic development, as evidenced by the early arrest caused by the knock-out mutation of URI1. With a hypomorphic uri1 allele, URI1 was shown to have a strong influence on the transcriptome. Consistent with this, the URI1 interactome shows that URI1 interacts with a relatively large number of partners, many of which are involved in fundamental processes related to mRNA metabolism. Thus, Arabidopsis URI1, like its orthologs in yeast and humans, appears to be involved in diverse cellular functions, including protein homeostasis, mRNA metabolism and signal transduction. URI is a highly versatile protein, although the molecular basis of this versatility is still unknown. Here we show that Arabidopsis URI1 possesses a large intrinsically disordered region spanning most of the C-terminal portion of the protein, a feature that is conserved in yeast and human orthologs. Our results reveal two main features of disordered proteins in URI1: promiscuity in interactions with partners and protein instability. We hypothesize that these two features contribute to endowing URI1 with functional versatility. Interestingly, the instability of URI1 is counteracted by sugar, and our genetic analysis places URI1 in the signaling pathway that controls growth in response to sugar deprivation-induced energy stress by acting as a negative upstream regulator of the master kinase TOR. We hypothesize that URI1 plays a role in preventing excessive seedling growth when energy conditions are favorable. / La realización de esta Tesis Doctoral ha sido posible gracias a una Ayuda para Contratos Predoctorales para la Formación de Doctores FPI (BES-2017-081041) y una ayuda europea EMBO Scientific Exchange (9583). Asímismo, el trabajo experimental ha sido financiado por el proyecto [ILOVEPFD] del Ministerio de Ciencia e Innovación AEI-MICINN (PID2019-109925GB-I00). / Gómez Mínguez, Y. (2024). Characterization of URI1 from Arabidopsis Thaliana and its role in stress responses [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203592

No description available.

Les papillomavirus sont des virus à ADN qui infectent la peau et les muqueuses. Ils causent des verrues et peuvent aussi mener au développement de cancers, dont le cancer du col de l’utérus. La réplication de leur génome nécessite deux protéines virales : l’hélicase E1 et le facteur de transcription E2, qui recrute E1 à l’origine de réplication virale. Pour faciliter l’étude de la réplication du génome viral, un essai quantitatif et à haut débit basé sur l’expression de la luciférase a été développé. Parallèlement, un domaine de transactivation a été identifié dans la région régulatrice N-terminale de la protéine E1. La caractérisation de ce domaine a montré que son intégrité est importante pour la réplication de l’ADN. Cette étude suggère que le domaine de transactivation de E1 est une région protéique intrinsèquement désordonnée qui permet la régulation de la réplication du génome viral par son interaction avec diverses protéines. / Papillomaviruses are small DNA viruses that infect skin and mucosa. They cause warts and can also lead to the development of cancers, including cervical cancer. Replication of their genome requires two viral proteins: the E1 helicase and the E2 transcription factor, which recruits E1 to the viral origin of replication. To facilitate the study of viral genome replication, a quantitative and high-throughput assay based on luciferase expression has been developed. In parallel, a transactivation domain has been identified in the N-terminal regulatory region of the E1 protein. Characterization of this domain showed that its integrity is important for DNA replication. This study suggests that the E1 transactivation domain is an intrinsically unstructured protein region that allows regulation of viral genome replication by its interaction with diverse proteins.

Les papillomavirus sont des virus à ADN qui infectent la peau et les muqueuses. Ils causent des verrues et peuvent aussi mener au développement de cancers, dont le cancer du col de l’utérus. La réplication de leur génome nécessite deux protéines virales : l’hélicase E1 et le facteur de transcription E2, qui recrute E1 à l’origine de réplication virale. Pour faciliter l’étude de la réplication du génome viral, un essai quantitatif et à haut débit basé sur l’expression de la luciférase a été développé. Parallèlement, un domaine de transactivation a été identifié dans la région régulatrice N-terminale de la protéine E1. La caractérisation de ce domaine a montré que son intégrité est importante pour la réplication de l’ADN. Cette étude suggère que le domaine de transactivation de E1 est une région protéique intrinsèquement désordonnée qui permet la régulation de la réplication du génome viral par son interaction avec diverses protéines. / Papillomaviruses are small DNA viruses that infect skin and mucosa. They cause warts and can also lead to the development of cancers, including cervical cancer. Replication of their genome requires two viral proteins: the E1 helicase and the E2 transcription factor, which recruits E1 to the viral origin of replication. To facilitate the study of viral genome replication, a quantitative and high-throughput assay based on luciferase expression has been developed. In parallel, a transactivation domain has been identified in the N-terminal regulatory region of the E1 protein. Characterization of this domain showed that its integrity is important for DNA replication. This study suggests that the E1 transactivation domain is an intrinsically unstructured protein region that allows regulation of viral genome replication by its interaction with diverse proteins.