Interactome of Ixr1, HMGB1 and HMGB2 proteins in relation to their cellular functions

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Abstract]The Saccharomyces cerevisiae protein, Ixr1, and human HMGB1 and HMGB2 proteins belong to the HMGB protein family. The main objective of this work was to identify new proteins binding Ixr1, HMGB1 and HMGB2 in order to increase the knowledge about their cellular functions. In yeast Ixr1 has activator or repressor activity on transcription in different promoters and controls the hypoxic response. The transcriptional activator domain of Ixr1 has been identified in its NH2 term in this study. Interactome studies show that Ixr1 binds to Ssn8 and this interaction might mediate the repressor function of Ixr1 on several promoters. Other proteins identified share functions associated to glucose metabolism, stress, and ribosome biogenesis among others. Differential phosphorylation of Ixr1 was observed depending on oxygen availability. The HMGB1 and HMGB2 interactomes were studied using different techniques as yeast two‐hybrid assays and purification methods coupled with mass spectrometry. This study was carried out in cancerous and noncancerous ovarian and prostate epithelial cells. Results showed the wide range of functions linked to their interacting partners. The interaction of HMGB1 with KRT7 and RBBP7 was validated by complementary techniques. HMGB1 specific interactions associated to cisplatin treatment of these cells were also identified in this study.[Resumen] La proteína de Saccharomyces cerevisiae Ixr1, y las proteínas humanas HMGB1 y HMGB2 se engloban dentro de la familia de proteínas HMGB. El principal objetivo de este trabajo ha sido la identificación de proteínas no previamente descritas que se unen a Ixr1, HMGB1 y HMGB2, para así poder ampliar el conocimiento sobre las funciones celulares de las proteínas HMGB. En levaduras, Ixr1 tiene un papel activador o represor de la transcripción sobre distintos promotores y controla la respuesta a hipoxia. El dominio de activación transcripcional de Ixr1 ha sido localizado en su extremo amino en este estudio. Ixr1 se une con Ssn8 y esta interacción puede mediar su papel represor sobre los promotores de algunos genes. Ixr1 además interacciona con proteínas relacionadas con el metabolismo de glucosa, estrés y biogénesis de ribosomas entre otras. Se ha detectado que Ixr1 presenta una fosforilación diferencial en función de la disponibilidad de oxígeno. Se ha llevado a cabo un estudio del interactoma de HMGB1 y HMGB2 mediante técnicas como el sistema de doble híbrido o purificaciones acopladas a espectrometría de masas en células epiteliales de ovario y próstata tumorales y no tumorales. Los resultados obtenidos ponen de manifiesto la amplia variedad de funciones asociadas a las proteínas que interactúan con HMGB1 o HMGB2. Se ha validado la interacción de HMGB1 con KRT7 y RBBP7 mediante otras técnicas complementarias. Así como también se han identificado interacciones específicas de HMGB1 en células tratadas con cisplatino.[Resumo] A proteína de Saccharomyces cerevisiae Ixr1, e as proteínas humanas HMGB1 e HMGB2 englóbanse dentro da familia de proteínas HMGB. O principal obxectivo deste traballo foi a identificación de proteínas non previamente descritas que se unen a Ixr1, HMGB1 e HMGB2, para así poder ampliar o coñecemento sobre as funcións celulares das proteínas HMGB. En lévedos, Ixr1 ten un papel activador ou represor da transcrición sobre distintos promotores e controla a resposta a hipoxia. O dominio de activación transcripcional de Ixr1 foi localizado no seu extremo amino neste estudo. Ixr1 únese con Ssn8 e esta interacción pode mediar o seu papel represor sobre os promotores dalgúns xenes. Ixr1 ademais, interacciona con proteínas relacionadas co metabolismo de glicosa, stress e ribosomais entre outras. Detectouse que Ixr1 presenta unha fosforilación diferencial en función da dispoñibilidade de osíxeno. Levouse a cabo un estudo do interactoma de HMGB1 e HMGB2 mediante técnicas como o sistema de dobre híbrido ou purificacións e espectrometría de masas en células epiteliais de ovario e próstata tumorais e non tumorais. Os resultados obtidos poñen de manifesto a ampla variedade de funcións asociadas ás proteínas que interactúan con HMGB1 ou HMGB2. Hase validado a interacción de HMGB1 con KRT7 e RBBP7 mediante outras técnicas complementarias. Así como tamén se identificaron interaccións específicas de HMGB1 en células tratadas con cisplatin

Aprendizaje cooperativo para el estudio semipresencial de técnicas de secuenciación de nueva generación

[Resumen] El aprendizaje cooperativo presenta un nuevo enfoque metodológico que pretende incrementar el rendimiento académico potenciando las relaciones sociales y el trabajo en grupo. En este trabajo, se ha diseñado el aprendizaje cooperativo para el estudio de las técnicas de secuenciación de nueva generación en la materia de génomica y proteómica del máster interuniversitario de biotecnología avanzada. Al finalizar la actividad, los alumnos deberán conocer los distintos tipos de secuenciaciones de nueva generación, describir en qué principios se basa cada uno y compararlos señalando sus ventajas e inconvenientes. Asimismo serán capaces de diseñar un experimento, en el que se pretenda secuenciar la totalidad del genoma de un organismo, utilizando una de estas técnicas. Todos los trabajos se expondrán oralmente y, como criterios de evaluación, el examen de la asignatura incluirá varias preguntas relacionadas con los trabajos de los alumnoshttps://doi.org/10.17979/spudc.978849749712

The Challenges and Opportunities of lncRNAs in Ovarian Cancer Research and Clinical Use

[Abstract] Ovarian cancer is one of the most lethal gynecological malignancies worldwide because it tends to be detected late, when the disease has already spread, and prognosis is poor. In this review we aim to highlight the importance of long non-coding RNAs (lncRNAs) in diagnosis, prognosis and treatment choice, to make progress towards increasingly personalized medicine in this malignancy. We review the effects of lncRNAs associated with ovarian cancer in the context of cancer hallmarks. We also discuss the molecular mechanisms by which lncRNAs become involved in cellular physiology; the onset, development and progression of ovarian cancer; and lncRNAs’ regulatory mechanisms at the transcriptional, post-transcriptional and post-translational stages of gene expression. Finally, we compile a series of online resources useful for the study of lncRNAs, especially in the context of ovarian cancer. Future work required in the field is also discussed along with some concluding remarks.This work was funded by Plan Estatal I + D + I by the Instituto de Salud Carlos III (ISCIII, Spain) under grant agreement AES number PI18/01714, cofounded by Fondo Europeo de Desarrollo Regional-FEDER (The European Regional Development Fund-ERDF) “A way of Making Europe,” and by Xunta de Galicia (Consolidación Grupos Referencia Competitiva contract number ED431C 2016-012). M.S.M. was funded by a predoctoral fellowship from FPU-2018 (Spain)Xunta de Galicia; ED431C 2016-01

Thanksgiving to Yeast, the HMGB Proteins History from Yeast to Cancer [Review]

This article belongs to the Special Issue Yeasts Biochemistry and Biotechnology[Abstract] Yeasts have been a part of human life since ancient times in the fermentation of many natural products used for food. In addition, in the 20th century, they became powerful tools to elucidate the functions of eukaryotic cells as soon as the techniques of molecular biology developed. Our molecular understandings of metabolism, cellular transport, DNA repair, gene expression and regulation, and the cell division cycle have all been obtained through biochemistry and genetic analysis using different yeasts. In this review, we summarize the role that yeasts have had in biological discoveries, the use of yeasts as biological tools, as well as past and on-going research projects on HMGB proteins along the way from yeast to cancer.This research was funded by Plan Estatal I+D+i, Instituto de Salud Carlos III (ISCIII, Spain) (grant nos. PI14/01031 and PI18/01417) and Ministerio de Ciencia e Innovación (grant no. PID2021-124564OB-I00), and cofunded by the Fondo Europeo de Desarrollo Regional-FEDER (The European Regional Development Fund-ERDF) “A way of Making Europe” and by Xunta de Galicia (Consolidación Grupos Referencia Competitiva grant no. ED431C 2020-08)Xunta de Galicia; ED431C 2020-0

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes

[Abstract] This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.This research was funded by the Wellcome Trust (grant no. 206194/Z/17/Z) and by Plan Estatal I+D+i, Instituto Carlos III (ISCIII, Spain) (grants no. PI14/01031 and PI18/01417) cofunded by the Fondo Europeo de Desarrollo Regional-FEDER (The European Regional Development Fund-ERDF) “A way of Making Europe”, and by Xunta de Galicia (Consolidación Grupos Referencia Competitiva grant no. ED431C 2020-08)Reino Unido. Wellcome Trust; 206194/Z/17/ZXunta de Galicia; ED431C 2020-0

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis

[Abstract] In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.This work has been funded by the contract ED431C 2020–08 from Xunta de Galicia (Consolidación Grupos Referencia Competitiva). A.R.-D. was funded by a research fellowship from Diputacion da Coruña-2017 (Spain)Xunta de Galicia; ED431C 2020–0

The HMGB1-2 Ovarian Cancer Interactome: the Role of HMGB Proteins and Their Interacting Partners MIEN1 and NOP53 in Ovary Cancer and Drug-Response

[Abstract] High mobility group box B (HMGB) proteins are overexpressed in different types of cancers such as epithelial ovarian cancers (EOC). We have determined the first interactome of HMGB1 and HMGB2 in epithelial ovarian cancer (the EOC-HMGB interactome). Libraries from the SKOV-3 cell line and a primary transitional cell carcinoma (TCC) ovarian tumor were tested by the Yeast Two Hybrid (Y2H) approach. The interactome reveals proteins that are related to cancer hallmarks and their expression is altered in EOC. Moreover, some of these proteins have been associated to survival and prognosis of patients. The interaction of MIEN1 and NOP53 with HMGB2 has been validated by co-immunoprecipitation in SKOV-3 and PEO1 cell lines. SKOV-3 cells were treated with different anti-tumoral drugs to evaluate changes in HMGB1, HMGB2, MIEN1 and NOP53 gene expression. Results show that combined treatment of paclitaxel and carboplatin induces a stronger down-regulation of these genes in comparison to individual treatments. Individual treatment with paclitaxel or olaparib up-regulates NOP53, which is expressed at lower levels in EOC than in non-cancerous cells. On the other hand, bevacizumab diminishes the expression of HMGB2 and NOP53. This study also shows that silencing of these genes affects cell-viability after drug exposure. HMGB1 silencing causes loss of response to paclitaxel, whereas silencing of HMGB2 slightly increases sensitivity to olaparib. Silencing of either HMGB1 or HMGB2 increases sensitivity to carboplatin. Lastly, a moderate loss of response to bevacizumab is observed when NOP53 is silenced.This work has been funded by the Projects Nº PI14/01031 and PI18/01714, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2013–2016 of the ISCIII- General Subdirection of Assesment and Promotion of the Research – European Regional Development Fund (FEDER) “A way of making Europe”. Funding is also acknowledged from Xunta de Galicia (Consolidación Grupos Referencia Competitiva Contract no. ED431C 2016–012). Aida Barreiro-Alonso was funded by a predoctoral fellowship from Xunta de Galicia-2013 (Spain) cofinanced by FEDERXunta de Galicia; ED431C 2016–01

O programa STEmbach na Área de Bioquímica e Bioloxía Molecular da Facultade de Ciencias

Colección: Contextos Universitarios Transformadores (CUT). Número 7[Resumo] O programa do bacharelato de excelencia en Ciencias e Tecnoloxía, STEMbach, é un programa da Xunta de Galicia, orientado a alumnos de primeiro e segundo de Bacharelato cuxo obxetivo é fomentar as ciencias entre os estudantes, baseándose no pensamento científico e a través dun plantexamento eminentemente práctico. O papel da Universidade e brindar aos alumnos a posibilidade de realizar proxectos científicos nas instalacións da UDC dirixidos por Profesores especialistas nas diversas materias, achegándoos, dun modo realista, ao mundo da investigación científica. A Área de Bioquímica e Bioloxía Molecular da Facultade de Ciencias participou nas tres últimas edicións, desenvolvendo seis proxectos distintos onde participaron catro IES, públicos e concertados e un total de 14 alumnos. Aínda que polo momento o número de estudantes que participaron nos proxectos STEMbach é relativamente baixo, as enquisas realizadas mostran que para a maioría o programa é útil e interesante, sobre todo a parte experimental realizada na UDC, destacando que lles gustaría dedicarlle máis tempo. Pola sua banda, os profesores de bacharelato manifestaron a sua preferencia polo establecemento dunha colaboración estable entre o seu centro e o proxecto da UDC seleccionado, repetindo o mesmo ano tras ano, e así poder axustar mellor a parte teórica á práctica.[Abstract] The program of the High School degree with excellence recognition in Science and Technology, STEMbach is a program of the Xunta de Galicia, aimed at first and second year high school students to promote science among students, based on scientific thinking and through an eminently practical approach. The role of the University is to provide students the possibility of carrying out scientific projects at UDC directed by specialist Professors in several subjects, bringing students closer to the world of scientific research in a realistic way. The Area of Biochemistry and Molecular Biology of the Faculty of Sciences has participated in the last three editions, developing six different projects where four IES, public and private and 14 students have participated. Although the number of students who participated in the STEMbach projects is relatively low, surveys show that for most students the program is useful and interesting, especially the experimental part performed at UDC, emphasizing that they would like to spend more time at laboraroy. On the other hand, the high school teachers expressed their preference for the establishment of a stable collaboration between their center and the selected UDC project, repeating the same project year after year, allowing adjust the theoretical part to practice.http://hdl.handle.net/2183/3249

HMGB Proteins from Yeast to Human. Gene Regulation, DNA Repair and Beyond

HMGB proteins are characterized for containing one or more HMG-box domains and are well conserved from yeasts to higher eukaryotes. The HMG-box domain is formed by three α-helices with an L-shaped fold. Although HMGB proteins also have cytoplasmic and extracellular functions, they bind to nuclear or mitochondrial DNA in a highly dynamic process that affects chromatin organization. In this review, we mainly focus on HMGB proteins from yeast and their human homologs as functionally involved in DNA repair and transcriptional regulation. Recent research reveals that these proteins participate in epigenetic control of gene expression, aging, disease, or stem-cell biology