Proteome-wide analyses og human stem cell differentiation using quantitative mass spectrometry and bioinformatics

Inigo Barrio-Hernandez

Proteome-wide analyses og human stem cell differentiation using quantitative mass spectrometry and bioinformatics

Inigo Barrio-Hernandez

Institut for Biokemi og Molekylær Biologi

Publikation: Afhandling › Ph.d.-afhandling

Abstract

Udviklingen fra encellede til mere komplekse multicellulære organismer medbragte den fordel at have celler som er fuldt ud specialiserede til én bestemt funktion. Denne specialisering kommer på bekostning af evnen til at undergå celledeling, hvilket nødvendiggør en population af mindre specialiserede celler kaldet stamceller. Celledifferentiering er den proces hvormed en celle går fra stamcelle til specialiseret celletype, og dette er blevet et vigtigt forskningsområde indenfor adskillige felter, eksempelvis regenerativ medicin. Specialiseringen involverer en stor forandring i proteinekspression og i cellesignaleringer, initieret af proteinfosforylering, hvilket gør det til et ideelt område at studere ved hjælp af massespektrometi-baseret kvantitativ proteomics og fosfoproteomics. Identificering of kvantificering af tusindvis af proteiner og modificeringer i eksperimenter af stort omfang skal dog kobles med computerbaserede analyser.

Arbejdet præsenteret i denne afhandling viser styrken af massespektrometri-baserede studier kombineret med bioinformatisk analyse til at udrede de molekylære mekanismer i celledifferentiering, i embryonisk og voksent organismeniveau. I artikel 1 fokuserede vi på den første dag i osteoblastdifferentiering. SILACbaseret kvantificering af fosforyleringer og proteiner fulgt af bioinformatisk analyse på forskellige niveauer tillod os at belyse signaleringsbegivenheder som orkestrerer denne proces. Vi var i stand til at forudsige og påvise PRKD1’s involvering i reguleringen af RunX2, samt andre vigtige signaleringsveje i osteoblastdifferentieringen. I artikel 2 opsatte vi et SILAC baseret kvantitativ forsøg til at studere human primær myoblastdifferentiering. Vi anvendte c-fuzzy clustering til at gruppere de forskelligt regulerede proteiner i forbindelse med muskeldifferentiering: cellecyklusstandsning, celleadhæsion, cellemigrering og myofibril formering. In silico transskriptionsfaktoranalyse komplementerede denne information, men var samtidig i stand til at følge proteiner potentielt involverede i myofibril udvikling samt muskelsygdomme; eksemplificerende styrken af vores model. I artikel 3 studerede vi rollen af MDM2 i de første skridt af adipocytdifferentiering. Vi benyttede to præ-adipocytiske mutantcellelinjer udtrykkende forskellige niveauer af MDM2 i et SILAC baseret assay, for at undersøge forskelle i proteinekspression. Transskriptionsfaktoranalyse viste STAT’s involvering, og opfølgende forsøg viste medieringen af CRTC transskriptionsfaktorer i forbindelse med reguleringen af adipocytdifferentiering. I artikel 4 studerede vi Pim2 kinasens rolle i hESC. SILAC baseret kvantitativ fosfoproteomicsstudier, koblet med Pim2 inhibitorer og tilfældig differentieringsinduktion, tillod os at bestemme en population af direkte og indirekte substrater som yderligere viser Pim2’s involvering i overgangen mellem selvfornyelse og differentiering.

Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Blagoev, Blagoy, Vejleder Kassem, Moustapha, Bivejleder
Udgivelsessted	Odense
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
Status	Udgivet - mar. 2016

Note vedr. afhandling

Afhandlingen kan læses på SDUs bibliotek. 

SDU link

Tjek adgangen til materialet i Syddansk Universitetsbiblioteks søgemaskine

2 Tidsskriftartikel

MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3
Hallenborg, P., Siersbæk, M., Barrio-Hernandez, I., Nielsen, R., Kristiansen, K., Mandrup, S., Grøntved, L. & Blagoev, B., 1. jul. 2016, I: Cell Death & Disease. 7, 6, s. e2289
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts
Le Bihan, M.-C., Barrio, I., Mortensen, T. P., Henningsen, J., Jensen, S. S., Bigot, A., Blagoev, B., Butler-Browne, G. & Kratchmarova, I., 7. aug. 2015, I: Journal of Proteome Research. 14, 8, s. 3348-3361
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Citationsformater

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title = "Proteome-wide analyses og human stem cell differentiation using quantitative mass spectrometry and bioinformatics",

abstract = "Evolution from unicellular to more complex multicellular organisms brought the advantage of having cells fully committed to one particular function. This specialization comes with loss of cell division capabilities making necessary the existence the existence of a pool of cells less specialized, called stem cells. Cell differentiation is the process of going from the second type to the first and it has become of great interest in several fields such as regenerative medicine. Stem cell commitment involves great changes in protein expression and furthermore in cell signalling events triggered by protein phosphorylation, what makes it a perfect area to apply mass spectrometry based quantitative proteomics and phosphoproteomics. However, identifying and quantifying thousands of features in large scale experiments need to be coupled with computational approaches.The work presented in this thesis shows the power of mass spectrometry based studies combined with bioinformatics analysis to unravel molecular mechanisms in cell differentiation, at embryonic and adult organism level. In Paper 1 we focused in the first day of osteoblastic differentiation. SILAC based quantitation of phosphosites and proteins followed by bioinformatics analysis at different levels allowed us to study the signalling events orchestrating this process. We were able to predict and prove PRKD1 implication in RunX2 regulation as well as other important pathways controlling osteoblastic lineage. In Paper 2 we set up a SILAC based quantitative approach to study human primary myoblast differentiation. We applied c-fuzzy clustering to group the regulated proteins following the different dynamic on muscle differentiation: cell cycle arrest, cell adhesion, migration and myofibril formation. Not only in silico transcription factor analysis complemented this information but also it was possible to track candidate proteins implicated in myofibril development and muscular diseases; demonstrating the strength of our model. In Paper 3, we studied the role of MDM2 in the first steps of adipocyte differentiation. Here, we used two pre-adipocyte mutant cell lines differing in Mdm2 expression and set up a SILAC based assay to see the difference in protein expression. Transcription factor analysis showed STATs implication and, subsequent follow ups the mediation of CRTC transcription factors in controlling adipocytic fate. In Paper 4 we studied Pim2 kinase role in hESC. SILAC based quantitative phosphoproteomic studies coupled with Pim2 inhibitors and random differentiation induction allowed to determine a pool of direct and indirect targets that further confirm Pim2 involvement in transition between self-renewal and differentiation.",

author = "Inigo Barrio-Hernandez",

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publisher = "Syddansk Universitet. Det Naturvidenskabelige Fakultet",

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AB - Evolution from unicellular to more complex multicellular organisms brought the advantage of having cells fully committed to one particular function. This specialization comes with loss of cell division capabilities making necessary the existence the existence of a pool of cells less specialized, called stem cells. Cell differentiation is the process of going from the second type to the first and it has become of great interest in several fields such as regenerative medicine. Stem cell commitment involves great changes in protein expression and furthermore in cell signalling events triggered by protein phosphorylation, what makes it a perfect area to apply mass spectrometry based quantitative proteomics and phosphoproteomics. However, identifying and quantifying thousands of features in large scale experiments need to be coupled with computational approaches.The work presented in this thesis shows the power of mass spectrometry based studies combined with bioinformatics analysis to unravel molecular mechanisms in cell differentiation, at embryonic and adult organism level. In Paper 1 we focused in the first day of osteoblastic differentiation. SILAC based quantitation of phosphosites and proteins followed by bioinformatics analysis at different levels allowed us to study the signalling events orchestrating this process. We were able to predict and prove PRKD1 implication in RunX2 regulation as well as other important pathways controlling osteoblastic lineage. In Paper 2 we set up a SILAC based quantitative approach to study human primary myoblast differentiation. We applied c-fuzzy clustering to group the regulated proteins following the different dynamic on muscle differentiation: cell cycle arrest, cell adhesion, migration and myofibril formation. Not only in silico transcription factor analysis complemented this information but also it was possible to track candidate proteins implicated in myofibril development and muscular diseases; demonstrating the strength of our model. In Paper 3, we studied the role of MDM2 in the first steps of adipocyte differentiation. Here, we used two pre-adipocyte mutant cell lines differing in Mdm2 expression and set up a SILAC based assay to see the difference in protein expression. Transcription factor analysis showed STATs implication and, subsequent follow ups the mediation of CRTC transcription factors in controlling adipocytic fate. In Paper 4 we studied Pim2 kinase role in hESC. SILAC based quantitative phosphoproteomic studies coupled with Pim2 inhibitors and random differentiation induction allowed to determine a pool of direct and indirect targets that further confirm Pim2 involvement in transition between self-renewal and differentiation.

M3 - Ph.D. thesis

PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet

CY - Odense

ER -

Proteome-wide analyses og human stem cell differentiation using quantitative mass spectrometry and bioinformatics

Abstract

Note vedr. afhandling

SDU link

Fingeraftryk

Relaterede publikationer

MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

Cellular Proteome Dynamics during Differentiation of Human Primary Myoblasts

Citationsformater