13,082 research outputs found
Protein quality control and aggregation in the endoplasmic reticulum: From basic to bedside
Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases
Sanfilippo syndrome: molecular basis, disease models and therapeutic approaches
Sanfilippo syndrome or mucopolysaccharidosis III is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. The exact molecular and cellular mechanisms by which impaired degradation and storage lead to cellular dysfunction and neuronal degeneration are still not fully understood. Here, we compile the knowledge on this issue and review all available animal and cellular models that can be used to contribute to increase our understanding of Sanfilippo syndrome disease mechanisms. Moreover, we provide an update in advances regarding the different and most successful therapeutic approaches that are currently under study to treat Sanfilippo syndrome patients and discuss the potential of new tools such as induced pluripotent stem cells to be used for disease modeling and therapy development
Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico
Valve replacement remains as the standard therapeutic option for aortic
stenosis patients, aiming at abolishing pressure overload and triggering
myocardial reverse remodeling. However, despite the instant hemodynamic
benefit, not all patients show complete regression of myocardial hypertrophy,
being at higher risk for adverse outcomes, such as heart failure. The current
comprehension of the biological mechanisms underlying an incomplete reverse
remodeling is far from complete. Furthermore, definitive prognostic tools and
ancillary therapies to improve the outcome of the patients undergoing valve
replacement are missing. To help abridge these gaps, a combined myocardial
(phospho)proteomics and pericardial fluid proteomics approach was followed,
taking advantage of human biopsies and pericardial fluid collected during
surgery and whose origin anticipated a wealth of molecular information
contained therein.
From over 1800 and 750 proteins identified, respectively, in the myocardium
and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated
proteins were detected. Gene annotation and pathway enrichment analyses,
together with discriminant analysis, are compatible with a scenario of increased
pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by
complement activity and other extrinsic factors, such as death receptor
activators), acute-phase response, immune system activation and fibrosis.
Specific validation of some targets through immunoblot techniques and
correlation with clinical data pointed to complement C3 β chain, Muscle Ring
Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation
regulated kinase 1A (DYRK1A) as potential markers of an incomplete
response. In addition, kinase prediction from phosphoproteome data suggests
that the modulation of casein kinase 2, the family of IκB kinases, glycogen
synthase kinase 3 and DYRK1A may help improve the outcome of patients
undergoing valve replacement. Particularly, functional studies with DYRK1A+/-
cardiomyocytes show that this kinase may be an important target to treat
cardiac dysfunction, provided that mutant cells presented a different response
to stretch and reduced ability to develop force (active tension).
This study opens many avenues in post-aortic valve replacement reverse
remodeling research. In the future, gain-of-function and/or loss-of-function
studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic
targets. Besides, clinical studies in larger cohorts will bring definitive proof of
complement C3, MuRF1 and DYRK1A prognostic value.A substituição da válvula aórtica continua a ser a opção terapêutica de
referência para doentes com estenose aórtica e visa a eliminação da
sobrecarga de pressão, desencadeando a remodelagem reversa miocárdica.
Contudo, apesar do benefício hemodinâmico imediato, nem todos os pacientes
apresentam regressão completa da hipertrofia do miocárdio, ficando com maior
risco de eventos adversos, como a insuficiência cardíaca. Atualmente, os
mecanismos biológicos subjacentes a uma remodelagem reversa incompleta
ainda não são claros. Além disso, não dispomos de ferramentas de
prognóstico definitivos nem de terapias auxiliares para melhorar a condição
dos pacientes indicados para substituição da válvula. Para ajudar a resolver
estas lacunas, uma abordagem combinada de (fosfo)proteómica e proteómica
para a caracterização, respetivamente, do miocárdio e do líquido pericárdico
foi seguida, tomando partido de biópsias e líquidos pericárdicos recolhidos em
ambiente cirúrgico.
Das mais de 1800 e 750 proteínas identificadas, respetivamente, no miocárdio
e no líquido pericárdico dos pacientes com estenose aórtica, um total de 90
proteínas desreguladas foram detetadas. As análises de anotação de genes,
de enriquecimento de vias celulares e discriminativa corroboram um cenário de
aumento da expressão de genes pro-hipertróficos e de síntese proteica, um
sistema ubiquitina-proteassoma ineficiente, uma tendência para morte celular
(potencialmente acelerada pela atividade do complemento e por outros fatores
extrínsecos que ativam death receptors), com ativação da resposta de fase
aguda e do sistema imune, assim como da fibrose.
A validação de alguns alvos específicos através de immunoblot e correlação
com dados clínicos apontou para a cadeia β do complemento C3, a Muscle
Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation
regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta
incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma,
sugere que a modulação da caseína cinase 2, a família de cinases do IκB, a
glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição
dos pacientes indicados para intervenção. Em particular, a avaliação funcional
de cardiomiócitos DYRK1A+/- mostraram que esta cinase pode ser um alvo
importante para tratar a disfunção cardíaca, uma vez que os miócitos mutantes
responderam de forma diferente ao estiramento e mostraram uma menor
capacidade para desenvolver força (tensão ativa).
Este estudo levanta várias hipóteses na investigação da remodelagem reversa.
No futuro, estudos de ganho e/ou perda de função realizados em
cardiomiócitos isolados ou em modelos animais de banding-debanding da
aorta ajudarão a testar a eficácia de modular os potenciais alvos terapêuticos
encontrados. Além disso, estudos clínicos em coortes de maior dimensão
trarão conclusões definitivas quanto ao valor de prognóstico do complemento
C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin
Towards personalized immunotherapy : development of in vitro models for imaging natural killer cell behavior in the tumor microenvironment
Tremendous advances in the tumor immunology field have transformed immunotherapy
from a promising approach to a standard clinical practice. However, a subset of cancer
patients is non-responsive to immunotherapy. More research is therefore needed to
understand the mechanisms underlying tumor resistance to immunotherapeutic treatments.
The aim of this doctoral work was to develop new tools to study the mechanisms of cancer
immunosurveillance and to test immunotherapeutic treatments in vitro. In this thesis, I
describe the methods developed, and I discuss the main biological findings obtained by
using these methods.
The thesis is organized as follows. A short historical background of immunotherapy is
provided in Chapter 1. Chapter 2 describes the principles of NK cell-mediated cancer
immunosurveillance, and provides an overview on rare cancers, mainly focusing on
sarcoma. The research aims are listed in Chapter 3. In Chapter 4, I describe the cell culture
methods and cell analysis techniques relevant for my doctoral work. In Chapter 5, I
describe the methods we developed to culture tumor spheroids in vitro using ultrasonic
standing waves in microwell chips, focusing on the theory, design, and applications.
Chapter 6 and Chapter 7 focus on the biological findings obtained using our platform in
combination with traditional immunological methods, followed by future implementations
discussed in Chapter 8. The constituent papers are provided at the end of the thesis.
In Paper I, we combined the use of the microwell chip, ultrasonic standing waves and a
protein-repellent polymer coating to enable the production of spheroids from multiple cell
types. In absence of cell adhesion to the chip, spheroids could be collected and further
analyzed by off-the-chip techniques.
In Paper II, we designed a novel multichambered microwell chip to perform multiplexed
fluorescence screening of two- or three-dimensional cell cultures. The platform allows the
direct assessment of drug or immune cell cytotoxic efficacy, making it a promising tool for
individualized cytotoxicity tests for personalized medicine.
In Paper III, we investigate the function of PVR receptors in NK cells interacting with
renal carcinoma spheroids, and the impact of PVR in NK cell-based cellular
immunotherapy. We demonstrated that variations in PVR expression are primarily
recognized by the inhibitory receptor TIGIT, while DNAM-1 strongly contributes to NK
cell activation mainly through PVR-independent mechanisms. We performed NK
cell-based cytotoxicity assays against renal carcinoma spheroids in the microwell chip.
Anti-TIGIT treatment was effective only for TIGIThigh NK cells both when used as
monotherapy or in combination with other drugs, suggesting that only a fraction of patients
might respond to anti-TIGIT therapy.
In Paper IV, a similar approach was used with primary sarcomas. We cultured
patient-derived sarcoma spheroids and tested NK cell-based immunotherapy in the
microwell chip, either alone or in combination with antibody therapy, and we identified
promising treatment combinations.
In Paper V, we applied the use of expansion microscopy to visualize NK cells infiltrating
renal carcinoma spheroids. In conclusion, our multi-disciplinary work shows the
development of new imaging-based platform and its use to study the mechanisms of NK
cell-mediated tumor surveillance and for personalized therapy
RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes
MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy.
HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD).
PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies.
In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD.
To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells.
To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression.
In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
A guide to designing photocontrol in proteins: methods, strategies and applications
Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique
Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro
The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human β- defensin (HBD)2 (by IL-1β) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVα2 and Laminin-β-1. Alternative pathways of protection initiated by IL-1β and TNFα stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context
Optimizing transcriptomics to study the evolutionary effect of FOXP2
The field of genomics was established with the sequencing of the human genome, a pivotal achievement that has allowed us to address various questions in biology from a unique perspective. One question in particular, that of the evolution of human speech, has gripped philosophers, evolutionary biologists, and now genomicists. However, little is known of the genetic basis that allowed humans to evolve the ability to speak. Of the few genes implicated in human speech, one of the most studied is FOXP2, which encodes for the transcription factor Forkhead box protein P2 (FOXP2). FOXP2 is essential for proper speech development and two mutations in the human lineage are believed to have contributed to the evolution of human speech. To address the effect of FOXP2 and investigate its evolutionary contribution to human speech, one can utilize the power of genomics, more specifically gene expression analysis via ribonucleic acid sequencing (RNA-seq).
To this end, I first contributed in developing mcSCRB-seq, a highly sensitive, powerful, and efficient single cell RNA-seq (scRNA-seq) protocol. Previously having emerged as a central method for studying cellular heterogeneity and identifying cellular processes, scRNA-seq was a powerful genomic tool but lacked the sensitivity and cost-efficiency of more established protocols. By systematically evaluating each step of the process, I helped find that the addition of polyethylene glycol increased sensitivity by enhancing the cDNA synthesis reaction. This, along with other optimizations resulted in developing a sensitive and flexible protocol that is cost-efficient and ideal in many research settings.
A primary motivation driving the extensive optimizations surrounding single cell transcriptomics has been the generation of cellular atlases, which aim to identify and characterize all of the cells in an organism. As such efforts are carried out in a variety of research groups using a number of different RNA-seq protocols, I contributed in an effort to benchmark and standardize scRNA-seq methods. This not only identified methods which may be ideal for the purpose of cell atlas creation, but also highlighted optimizations that could be integrated into existing protocols.
Using mcSCRB-seq as a foundation as well as the findings from the scRNA-seq benchmarking, I helped develop prime-seq, a sensitive, robust, and most importantly, affordable bulk RNA-seq protocol. Bulk RNA-seq was frequently overlooked during the efforts to optimize and establish single-cell techniques, even though the method is still extensively used in analyzing gene expression. Introducing early barcoding and reducing library generation costs kept prime-seq cost-efficient, but basing it off of single-cell methods ensured that it would be a sensitive and powerful technique. I helped verify this by benchmarking it against TruSeq generated data and then helped test the robustness by generating prime-seq libraries from over seventeen species. These optimizations resulted in a final protocol that is well suited for investigating gene expression in comprehensive and high-throughput studies.
Finally, I utilized prime-seq in order to develop a comprehensive gene expression atlas to study the function of FOXP2 and its role in speech evolution. I used previously generated mouse models: a knockout model containing one non-functional Foxp2 allele and a humanized model, which has a variant Foxp2 allele with two human-specific mutations. To study the effect globally across the mouse, I helped harvest eighteen tissues which were previously identified to express FOXP2. By then comparing the mouse models to wild-type mice, I helped highlight the importance of FOXP2 within lung development and the importance of the human variant allele in the brain.
Both mcSCRB-seq and prime-seq have already been used and published in numerous studies to address a variety of biological and biomedical questions. Additionally, my work on FOXP2 not only provides a thorough expression atlas, but also provides a detailed and cost-efficient plan for undertaking a similar study on other genes of interest. Lastly, the studies on FOXP2 done within this work, lay the foundation for future studies investigating the role of FOXP2 in modulating learning behavior, and thereby affecting human speech
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