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The skin has a system that can detect light in a fashion similar to the retina. Although its presence was initially reported almost 20 years ago, only in 2011 functional studies started to be reported. The biological clock of the skin has also been reported in the beginning of the century, but its function and relevance still remain unexplored. Thus, this Ph.D. project was designed to explore the functionality of both systems in melanocytes, and whether the disruption of these systems is associated with the development of melanoma cancer. Using in vitro, in vivo, and bioinformatics approaches, we have shown that: 1) the biological clock of malignant melanocytes is more responsive to visible light, UVA radiation, estradiol, and temperature compared to normal cells; 2) UVA radiation is detected by melanopsin (OPN4) and rhodopsin (OPN2), which triggers a cGMP related cascade that leads to immediate pigment darkening (IPD) in normal and malignant melanocytes; 3) in addition to detecting UVA radiation, OPN4 also senses thermal energy, which activates the biological clock of both normal and malignant melanocytes; 4) regarding the biological clock, we have provided several layers of evidence that proves that in melanoma a chronodisruption scenario is established compared to healthy skin and/or normal pigment cells; 5) in vivo tumor samples display a low amplitude circadian rhythm of clock gene expression and an ultradian oscillatory profile in melanin content; 6) a non-metastatic melanoma leads to a systemic chronodisruption, which we suggest that could favor the metastatic process; 7) in human melanoma, we demonstrated the role of BMAL1 as a prognostic marker and a putative marker of immune therapy success. Taken altogether, these results significantly contributed to the literature as it brought to light new and interesting targets and processes, which will be explored in future projects / A pele possui um sistema que pode detectar luz de forma análoga à retina. Embora a presença deste sistema tenha sido inicialmente descrita quase há 20 anos, apenas no ano de 2011 estudos funcionais começaram a ser relatados. Sabe-se que o relógio biológico da pele também foi identificado no início do século, mas sua função e relevância ainda continuam pouco exploradas. Diante deste cenário, este projeto de doutorado foi desenhado para investigar a funcionalidade de ambos os sistemas em melanócitos e se perturbação dos mesmos estaria associada com o desenvolvimento de melanoma. Através do uso de abordagens in vitro, in vivo e de bioinformática, nós demonstramos que: 1) o relógio biológico de melanócitos malignos é mais responsivo à luz visível, radiação UVA, estradiol e temperatura comparado ao de células normais; 2) a radiação UVA é detectada por melanopsina (OPN4) e rodopsina (OPN2), que ativam uma via de sinalização dependente de GMPc, levando ao processo de pigmentação imediata (IPD) em melanócitos normais e malignos; 3) além de detecção de radiação UVA, a OPN4 também detecta energia térmica que, por sua vez, ativa o relógio biológico de melanócitos normais e malignos; 4) relativo ao relógio biológico, provamos por diferentes abordagens que, no melanoma, um cenário de cronoruputura está estabelecido em comparação a pele saudável e/ou melanócitos; 5) tumores in vivo apresentam um ritmo circadiano de baixa amplitude na expressão dos genes de relógio e um ritmo ultradiano oscilatório no conteúdo de melanina; 6) um melanoma não metastático leva a um quadro sistêmico de cronoruptura, o qual sugerimos favorecer o processo de metástase; 7) em melanoma humano, demonstramos o papel do gene BMAL11 como marcador de prognóstico e um possível indicador de sucesso de imunoterapias. Portanto, este projeto contribuiu de forma significante para a literatura científica uma vez que trouxe à luz novos e interessantes alvos terapêuticos e processos, os quais serão explorados em projetos futuros

Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps-ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A-B and helices C-G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.

Interactions between proteins give rise to many functions in cells. In the lastdecade, highthroughput experiments have identified thousands of protein interactions, which are often represented together as large protein interaction networks. However, the classical way of representing interaction networks, as nodes and edges, is too limited to take dynamic properties such as compatible and mutually exclusive interactions into account. In this work, we study protein interaction networks using structural information. More specifically, the analysis of protein interfaces in threedimensional protein structures enables us to identify which interfaces are compatible and which are not. Based on this principle, we have implemented a method, which aims at the analysis of protein interaction networks from a structural point of view by (1) predicting possible binary interactions for proteins that have been found in complex experimentally and (2) identifying possible mutually exclusive and compatible complexes. We validated our method by using positive and negative reference sets from literature and set up an assay to benchmark the identification of compatible and mutually exclusive structural interactions. In addition, we reconstructed the protein interaction network associated with the G proteincoupled receptor Rhodopsin and defined related functional submodules by combining interaction data with structural analysis of the network. Besides its established role in vision, our results suggest that Rhodopsin triggers two additional signaling pathways towards (1) cytoskeleton dynamics and (2) vesicular trafficking. / Las funciones de las proteínas resultan de la manera con la que interaccionan entre ellas. Los experimentos de alto rendimiento han permitido identificar miles de interacciones de proteínas que forman parte de redes grandes y complejas. En esta tesis, utilizamos la información de estructuras de proteínas para estudiar las redes de interacciones de proteínas. Con esta información, se puede entender como las proteínas interaccionan al nivel molecular y con este conocimiento se puede identificar las interacciones que pueden ocurrir al mismo tiempo de las que están incompatibles. En base a este principio, hemos desarrollado un método que permite estudiar las redes de interacciones de proteínas con un punto de vista mas dinámico de lo que ofrecen clásicamente. Además, al combinar este método con minería de la literatura y Los datos de la proteomica hemos construido la red de interacciones de proteínas asociada con la Rodopsina, un receptor acoplado a proteínas G y hemos identificado sus sub--‐módulos funcionales. Estos análisis surgieron una novel vıa de señalización hacia la regulación del citoesqueleto y el trafico vesicular por Rodopsina, además de su papel establecido en la visión.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 6 uppsatser.

High visual sensitivity is a common but important characteristic of animal eyes. It is especially critical for night vision. In animal eyes, photoreceptors are the first to receive the incoming rays of light and they convert the light signals to electrical signals before passing the information to interneurons in the eye and finally to the brain. To function in dim light conditions, photoreceptors have developed high sensitivities to light. It is reported that both mammalian rod photoreceptors and Drosophilaphotoreceptors can detect single photons. The high sensitivities of photoreceptors largely depend on a high content of rhodopsin, a light-stimulated G protein-coupled receptor (GPCR), in light sensory organelles, outer segments in mammals and rhabdomeres in Drosophila. Two shared characteristics, the tightly packed photoreceptive membrane and the high concentration of rhodopsin in the membrane, work together to enable the photoreceptors to achieve the high content of rhodopsin in photosensory organelles in both mammals and Drosophila. In this thesis, I have used the Drosophilaeye as a model system to study the molecular mechanisms required for the maintenance of these two characteristics. In the second chapter, I present a new molecular mechanism of preventing Gq-mediated rhabdomeral degeneration. A new gene named tadr (for torn and diminished rhabdomeres), when mutated, leads to visual sensitivity reduction and photoreceptor degeneration. Degeneration in the tadr mutant is characterized by shrunken and disrupted rhabdomeres. The TADR protein interacts in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppresses the tadr-induced degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 fail to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses reveal that, in the tadr mutant, the Gq protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of Gq level by introducing a hypomorphic allele of Gαq gene greatly inhibits the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the Gqsignaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans. In the third chapter, I present a Drosophila CUB- and LDLa-domain transmembrane protein CULD that counteracts the visual arrestin Arr1-mediated endocytosis to retain rhodopsin in rhabdomeral membrane. CULD is mostly localized in rhabdomeres, but is also detected in scarce rhodopsin endocytic vesicles that contain Arr1. An intracellular region of CULD interacts with Arr1 in vitro. In both culdmutant and knockdown flies, a large amount of rhodopsin is mislocalized in the cell body of photoreceptors through lightdependent, Arr1-mediated endocytosis, leading to reduction of photoreceptor sensitivity. Expressing a wild-type CULD protein in photoreceptors, but not a mutant variant lacking the Arr1-interacting site, rescues both the rhodopsin mislocalization and the low sensitivity phenotypes. Once rhodopsin has been internalized in adult mutant flies, it is reversed only by expression of CULD but not by blocking endocytosis, suggesting that CULD promotes recycling of endocytosed rhodopsin to the rhabdomere. Our results demonstrate an important role of CULD in the maintenance of membrane rhodopsin density and photoreceptor sensitivity. We propose that a common cellular function of CUB- and LDLa-domain proteins, in both mammals and invertebrates, is to concentrate receptors including GPCRs in particular regions of cell membrane. In summary, the work addressed in this thesis has identified new molecular mechavii nisms underlying the maintenance of visual sensitivity in Drosophila, either through preventing Gq-mediated rhabdomeral degeneration or through antagonizing arrestin-mediated rhodopsin endocytosis. This work has advanced our understanding of visual biology and the general regulatory mechanisms of GPCR signaling, and may provide valuable clues to pathologic studies of human retinal degeneration disorders.

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