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As distrofias musculares (DM) se caracterizam por degeneração progressiva e irreversível da musculatura esquelética. Dentre estas, a Distrofia Muscular de Duchenne (DMD), constitui doença letal ligada ao cromossomo X, caracterizada pela ausência de distrofina na membrana das fibras musculares, afetando um em cada 3.000 meninos nascidos vivos, sendo que a maioria será confinada à cadeira de rodas a partir dos 6 anos de idade e com sobrevida, sem cuidados especiais, em torno dos 25. Apesar do uso de técnicas modernas de diagnóstico, ainda não existe tratamento eficaz para essas doenças. Alguns trabalhos das décadas de 70 e 80 sugeriram que a inibição do hormônio do crescimento (GH) poderia retardar a evolução do processo distrófico de pacientes com DMD. Entretanto, como essas observações não foram comprovadas cientificamente até a presente data, neste trabalho estudamos os efeitos do excesso e da inibição do GH na evolução do quadro distrófico em modelo murino de distrofia muscular: i) administrou-se GH (10μg/ camundongo/dia, i.p., por 10 dias) em camundongos: a) distróficos dy/dy (B6.Wk-Lama2dy-2J/J) com fenótipo, resultando na aceleração dos sintomas da doença, traduzida pela piora das performances de 17% no teste de deambulação (TD), 64% no teste de sustentação (TS), 28% no teste da esteira (TE) e 55% no teste de força máxima (TFM), em relação aos distróficos que não tomaram GH; apenas no teste de resistência à inclinação (TRI) não houve diferença significativa; entretanto, cortes histológicos do músculo gastrocnêmico desses camundongos, não mostraram alterações significativas em relação aos distróficos que não tomaram a droga; quanto aos marcadores de degeneração e regeneração (MDR) na musculatura desses animais, o GH não alterou os níveis de Pax7, MyoD, Laminina e Desmina, mas aumentou de 90% os níveis de TGFβ-1, uma citocina inflamatória que induz fibrose; b) já nos camundongos normais, o GH, ao contrário dos resultados acima, aumentou as performances de 13% no TD e de 28% TS, e ligeiramente, de forma não significativa, em outros dois testes; cortes histológicos revelaram que o GH aumentou de 16,5% a área de secção transversal dos músculos desses animais, o que poderia explicar os resultados descritos acima; já os níveis dos MDR, incluindo o de TGFβ-1, estavam semelhantes aos dos animais controles; ii) desenvolveu-se um novo modelo experimental resultante de cruzamentos de camundongos geneticamente deficientes em GH (Ghrhrlit) com os distróficos dy/dy (B6.Wk-Lama2dy-2J/J), mostrando um retardo na evolução da doença: as performances dos anões distróficos em relação aos anões normais foram melhores que aquelas dos distróficos em relação aos animais normais, ou sejam, 14% maior TD, 71% maior no TS, 18% maior no TE, 5,5% maior no TRI e 102% maior no TFM; cortes histológicos mostraram que a área do interstício nos músculos dos anões distróficos foi 29% menor que a dos distróficos não anões; os níveis de TGFβ-1 nestes animais, foram os únicos entre aqueles dos MDR estudados, que encontravam-se diminuidos de 36% em relação àqueles dos distróficos não anões; iii) a inibição da liberação do GH pela octreatida, análogo da somatostatina, mostrou também uma relativa melhora ou uma estabilização na evolução do quadro distrófico, traduzido pelo aumento, após 60 dias de tratamento, das performances nos testes funcionais da musculatura em relação àquelas dos animais distróficos sem tratamento: foi 12% maior no TD enquanto os distróficos controles diminuíram em 14%; foi 27% maior no teste de Rota Rod, enquanto os distróficos controles diminuíram em 32%; mantiveram a mesma performance no TE, enquanto os distróficos controles pioraram em 23%, e foram 29% maiores no TS, e 43% no teste de força de preensão, enquanto os distróficos controles não melhoram as suas performances nestes testes. Esses resultados sugerem um efeito patológico do GH na musculatura distrófica, com seu excesso levando a uma piora da doença, possivelmente através do aumento dos níveis de TGFβ-1. A diminuição do GH, consequentemente, poderia levar a um retardo da evolução da doença. Estes estudos experimentais abrem perspectivas bastante promissoras para se testar inibição dos efeitos do GH na evolução das doenças distróficas humanas. / Muscular dystrophies (DM) are characterized by progressive and irrevertible degeneration of the skeleton muscles. Among them, Duchenne Muscular Dystrophy (DMD) consitutes lethal disease linked to chromossome X, distinguished by the absence of dystrophyn in the muscular fibers membrane, affecting one in each 3,000 boys born alive, who, most of them, will be confined to wheel chairs since the age of 6 and with life expectance, without special care, of 25 years. Despite modern diagnosis techniques, there is not an efficient treatment for these diseases. Some works from the 1970´s and 1980´s suggested that the inhibition of the growth hormone (GH) could retard the dystrophy process evolution in DMD patients. Nevertheless since this observation has not been proven scientifically, in the present work, the GH excess and inhibition effects on a murino model of muscular dystrophy, were studied: i) GH (10μg/mouse/day, i.p., for 10 days) was given to mice: a) dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), with phenotype , resulting in the acceleration of the disease symptoms, confirmed by the performance worsening by 17% in the deambulation test (DT), 64% in the sustaining test (S), 28% in the mat test and 55% in the maximum strength test (MST), in relation to dystrophic mice which had not taken GH; only in the inclination resistance test (IRT) there was no significant difference; however, histological cuts of the gastrocnemius muscle of these mice did not show significant alterations, comparing to dystrophic mice which had not taken the drug: as to degeneration and regeneration markers (DRM) in these animals muscles, the GH did not alter Pax7, MyoD. Laminin and Desmin levels, although it increased by 90% the TGFβ-1 levels, an inflammatory cytokine, that induces fibrosis b) in normal mice, the GH, opposite to the results above, increased the performances by 13% in the DT and by 28% in the ST, and slightly, not significantly, in other two tests; histological cuts revealed that the GH increased by 16.5% the transversal section area of these animals muscles, what could explain the results described above; as to the MDR levels, including those of the TGFβ-1, they were similar to the control animals; ii) a new experimental model, resulting from the crossing of GH genetically deficient mice (Ghrthlit) with dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), showing retardation in the disease evolution; the performances of the dystrophic dwarfs, compared to the normal dwarfs, were better than those of the dystrophic compared to normal animals, namely, 14% larger DT, 71% superior in the ST, 18% larger in the MT, 5.5% larger in the IRT and 102% superior in the MST; histological cuts showed that the interstice area in the dystrophic dwarfs muscles was 29% smaller than that of non-dwarfs dystrophic; the TGFβ-1 levels in these animals were the only MDR studied, which were found to be diminished by 36%, compared to those of the non-dwarfs dystrophic; the GH release inhibition by octreotide, somatostatin analogous, also showed a relative improvement or stabilization in the dystrophy evolution, demonstrated by the increase, ater 60 days treatment, of the performances in the muscle functional tests, compared to those of the dystrophic animals, without treatment: it was 12% larger in the DT, while the control dystrophic animals worsened by 32%; they maintained the same performance in the ST, and 43% in the seizing strength test, while the control dystrophic did not improve their performances in these tests. These results suggest a GH pathologic effect, possibly through the TGFβ-1 levels increase. The GH diminishing, consequently, could lead to the retardation in the disease evolution. These experimental studies open promissing perspectives to test the GH effects inhibition in human dystrophic diseases evolution.

As distrofias musculares (DM) se caracterizam por degeneração progressiva e irreversível da musculatura esquelética. Dentre estas, a Distrofia Muscular de Duchenne (DMD), constitui doença letal ligada ao cromossomo X, caracterizada pela ausência de distrofina na membrana das fibras musculares, afetando um em cada 3.000 meninos nascidos vivos, sendo que a maioria será confinada à cadeira de rodas a partir dos 6 anos de idade e com sobrevida, sem cuidados especiais, em torno dos 25. Apesar do uso de técnicas modernas de diagnóstico, ainda não existe tratamento eficaz para essas doenças. Alguns trabalhos das décadas de 70 e 80 sugeriram que a inibição do hormônio do crescimento (GH) poderia retardar a evolução do processo distrófico de pacientes com DMD. Entretanto, como essas observações não foram comprovadas cientificamente até a presente data, neste trabalho estudamos os efeitos do excesso e da inibição do GH na evolução do quadro distrófico em modelo murino de distrofia muscular: i) administrou-se GH (10μg/ camundongo/dia, i.p., por 10 dias) em camundongos: a) distróficos dy/dy (B6.Wk-Lama2dy-2J/J) com fenótipo, resultando na aceleração dos sintomas da doença, traduzida pela piora das performances de 17% no teste de deambulação (TD), 64% no teste de sustentação (TS), 28% no teste da esteira (TE) e 55% no teste de força máxima (TFM), em relação aos distróficos que não tomaram GH; apenas no teste de resistência à inclinação (TRI) não houve diferença significativa; entretanto, cortes histológicos do músculo gastrocnêmico desses camundongos, não mostraram alterações significativas em relação aos distróficos que não tomaram a droga; quanto aos marcadores de degeneração e regeneração (MDR) na musculatura desses animais, o GH não alterou os níveis de Pax7, MyoD, Laminina e Desmina, mas aumentou de 90% os níveis de TGFβ-1, uma citocina inflamatória que induz fibrose; b) já nos camundongos normais, o GH, ao contrário dos resultados acima, aumentou as performances de 13% no TD e de 28% TS, e ligeiramente, de forma não significativa, em outros dois testes; cortes histológicos revelaram que o GH aumentou de 16,5% a área de secção transversal dos músculos desses animais, o que poderia explicar os resultados descritos acima; já os níveis dos MDR, incluindo o de TGFβ-1, estavam semelhantes aos dos animais controles; ii) desenvolveu-se um novo modelo experimental resultante de cruzamentos de camundongos geneticamente deficientes em GH (Ghrhrlit) com os distróficos dy/dy (B6.Wk-Lama2dy-2J/J), mostrando um retardo na evolução da doença: as performances dos anões distróficos em relação aos anões normais foram melhores que aquelas dos distróficos em relação aos animais normais, ou sejam, 14% maior TD, 71% maior no TS, 18% maior no TE, 5,5% maior no TRI e 102% maior no TFM; cortes histológicos mostraram que a área do interstício nos músculos dos anões distróficos foi 29% menor que a dos distróficos não anões; os níveis de TGFβ-1 nestes animais, foram os únicos entre aqueles dos MDR estudados, que encontravam-se diminuidos de 36% em relação àqueles dos distróficos não anões; iii) a inibição da liberação do GH pela octreatida, análogo da somatostatina, mostrou também uma relativa melhora ou uma estabilização na evolução do quadro distrófico, traduzido pelo aumento, após 60 dias de tratamento, das performances nos testes funcionais da musculatura em relação àquelas dos animais distróficos sem tratamento: foi 12% maior no TD enquanto os distróficos controles diminuíram em 14%; foi 27% maior no teste de Rota Rod, enquanto os distróficos controles diminuíram em 32%; mantiveram a mesma performance no TE, enquanto os distróficos controles pioraram em 23%, e foram 29% maiores no TS, e 43% no teste de força de preensão, enquanto os distróficos controles não melhoram as suas performances nestes testes. Esses resultados sugerem um efeito patológico do GH na musculatura distrófica, com seu excesso levando a uma piora da doença, possivelmente através do aumento dos níveis de TGFβ-1. A diminuição do GH, consequentemente, poderia levar a um retardo da evolução da doença. Estes estudos experimentais abrem perspectivas bastante promissoras para se testar inibição dos efeitos do GH na evolução das doenças distróficas humanas. / Muscular dystrophies (DM) are characterized by progressive and irrevertible degeneration of the skeleton muscles. Among them, Duchenne Muscular Dystrophy (DMD) consitutes lethal disease linked to chromossome X, distinguished by the absence of dystrophyn in the muscular fibers membrane, affecting one in each 3,000 boys born alive, who, most of them, will be confined to wheel chairs since the age of 6 and with life expectance, without special care, of 25 years. Despite modern diagnosis techniques, there is not an efficient treatment for these diseases. Some works from the 1970´s and 1980´s suggested that the inhibition of the growth hormone (GH) could retard the dystrophy process evolution in DMD patients. Nevertheless since this observation has not been proven scientifically, in the present work, the GH excess and inhibition effects on a murino model of muscular dystrophy, were studied: i) GH (10μg/mouse/day, i.p., for 10 days) was given to mice: a) dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), with phenotype , resulting in the acceleration of the disease symptoms, confirmed by the performance worsening by 17% in the deambulation test (DT), 64% in the sustaining test (S), 28% in the mat test and 55% in the maximum strength test (MST), in relation to dystrophic mice which had not taken GH; only in the inclination resistance test (IRT) there was no significant difference; however, histological cuts of the gastrocnemius muscle of these mice did not show significant alterations, comparing to dystrophic mice which had not taken the drug: as to degeneration and regeneration markers (DRM) in these animals muscles, the GH did not alter Pax7, MyoD. Laminin and Desmin levels, although it increased by 90% the TGFβ-1 levels, an inflammatory cytokine, that induces fibrosis b) in normal mice, the GH, opposite to the results above, increased the performances by 13% in the DT and by 28% in the ST, and slightly, not significantly, in other two tests; histological cuts revealed that the GH increased by 16.5% the transversal section area of these animals muscles, what could explain the results described above; as to the MDR levels, including those of the TGFβ-1, they were similar to the control animals; ii) a new experimental model, resulting from the crossing of GH genetically deficient mice (Ghrthlit) with dystrophic dy/dy (B6.Wk-Lama2dy-2J/J), showing retardation in the disease evolution; the performances of the dystrophic dwarfs, compared to the normal dwarfs, were better than those of the dystrophic compared to normal animals, namely, 14% larger DT, 71% superior in the ST, 18% larger in the MT, 5.5% larger in the IRT and 102% superior in the MST; histological cuts showed that the interstice area in the dystrophic dwarfs muscles was 29% smaller than that of non-dwarfs dystrophic; the TGFβ-1 levels in these animals were the only MDR studied, which were found to be diminished by 36%, compared to those of the non-dwarfs dystrophic; the GH release inhibition by octreotide, somatostatin analogous, also showed a relative improvement or stabilization in the dystrophy evolution, demonstrated by the increase, ater 60 days treatment, of the performances in the muscle functional tests, compared to those of the dystrophic animals, without treatment: it was 12% larger in the DT, while the control dystrophic animals worsened by 32%; they maintained the same performance in the ST, and 43% in the seizing strength test, while the control dystrophic did not improve their performances in these tests. These results suggest a GH pathologic effect, possibly through the TGFβ-1 levels increase. The GH diminishing, consequently, could lead to the retardation in the disease evolution. These experimental studies open promissing perspectives to test the GH effects inhibition in human dystrophic diseases evolution.

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients. TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling. The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model. In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner. In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells. In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.

Le Spina Bifida est une maladie congénitale rare due à anomalie de fermeture du tube neural ayant des conséquences physiques et fonctionnelles multiples. La prise en charge des séquelles digestives du Spina Bifida ne fait l’objet d’aucun consensus national ou international. Les objectifs de cette thèse translationnelle étaient de caractériser les plaintes anorectales des patients adultes ayant un Spina Bifida, d’en préciser les mécanismes physiopathologiques sous-jacents à partir des données de la manométrie anorectale et du barostat rectal et en étudiant les fonctions de la barrière épithéliale intestinale. Les plaintes anorectales des patients adultes ayant un Spina Bifida représentent leur deuxième plainte en terme de fréquence et sont l’incontinence fécale, et la constipation. Ces troubles peuvent évoluer au cours du temps ce qui justifie un suivi digestif au long cours. Le niveau lésionnel neurologique n’est ni associé aux troubles digestifs ni à à leur évolution. Les patients ayant un Spina Bifida ont une atteintede la fonction anale (défaut de contraction) et des fonctions rectales (diminution du tonus et de la compliance rectale). Ils ont également une hyperperméabilité paracellulaire et une diminution de la densité du tissu conjonctif; ces 2 anomalies étant corrélées entre elles. L’augmentation de la perméabilité paracellulaire était négativement corrélée avec la compliance rectale. Enfin, les patients ayant un Spina Bifida ont une augmentation de l’expression de TGFBeta1. Les anomalies des fonctions anorectales et de la barrière épithéliale intestinale pourraient être des cibles thérapeutiques potentielles dans la prise en charge des troubles anorectaux des patients adultes ayant un Spina Bifida. / Spina Bifida is a rare congenital disorder caused by an abnormal neural tube closure with multiple physical and functional consequences. The management of the digestive disorders of Spina Bifida is not consensual. The objectives of this present work were to characterize the anorectal complaints of adult patients with Spina Bifida, to specify the underlying pathophysiological mechanisms from the data of anorectal manometry and rectal barostat and to study the functions of the intestinal epithelial barrier. The anorectal complaints of adult patients with Spina Bifida represent their second complaint and are faecal incontinence and constipation. These disorders can change over time which justifies long-term digestive follow-up. The level of neurological lesion is neither associated with digestive disorders nor with their evolution. Patients with Spina Bifida have impaired anal function (lack of contraction) and rectal function (decreased tone and rectal compliance). They also have paracellular hyperpermeability and decreased connective tissue density ; these 2 anomalies are interrelated. The increase in paracellular permeability was negatively correlated with rectal compliance. Finally, patients with Spina Bifida have an increased expression of TGFBeta1. Abnormalities of anorectal function and intestinal epithelial barrier may be potential therapeutic targets in the management of anorectal disorders in adult patients with Spina Bifida.

An der Interpretation extrazellulärer Signale beteiligte Regulationsnetzwerke sind von zentraler Bedeutung für alle Organismen. Extrazelluläre Signale werden gewöhnlich durch enzymatische Kaskaden innerhalb weniger Minuten in den Zellkern weitergeleitet, wo sie langsame Änderungen der Genexpression bewirken und so das Schicksal der Zelle beeinflussen. Im ersten Teil der Arbeit wird durch mathematische Modellierung untersucht, wie die MAPK Kaskade Signale von der Zellmembran in den Kern weiterleitet. Es wurden Netzwerkeigenschaften herausgearbeitet, die verhindern, dass die MAPK Kaskade fälschlicherweise durch genetische Mutationen aktiviert wird. Desweiteren wurde eine versteckte positive Rückkopplungsschleife identifiziert, welche die Aktivierung der MAPK Kaskade oberhalb eines gewissen Schwellwert-Stimulus verstärkt. Der zweite Teil der Arbeit konzentriert sich darauf, wie Änderungen der Genexpression auf langsamer Zeitskala in das Signalnetzwerk rückkoppeln. Eine systematische Genexpressionsdaten-Analyse ergab, dass transkriptionelle Rückkopplung in Eukaryoten generell über Induktion kurzlebiger Signalinhibitoren geschieht. Dynamische Modellierung und experimentelle Validierung von Modellvorhersagen ergab, dass das Inhibitorprotein SnoN als zentraler negativer Feedback Regulator im TGFbeta Signalweg fungiert. Der dritte Teil der Arbeit untersucht, wie die Genexpressionsmaschinerie intrazelluläre Signale interpretiert (“dekodiert“). Eine experimentelle und theoretische Analyse der cyanobakteriellen Eisenstress-Antwort ergab, dass IsrR, eine kleine regulatorische RNA, die Genexpression auf ausreichend starke und lange Stimulation beschränkt. Des Weiteren wurde ein “Reverse Engineering“-Algorithmus auf Hochdurchsatz-RNAi-Daten angewendet, um die Topologie eines krebsrelevanten Transkriptionsfaktornetzwerks abzuleiten. Zusammenfassend wurde in dieser Dissertation gezeigt, wie mathematische Modellierung die experimentelle Analyse biologischer Systeme unterstützen kann. / Intracellular regulatory networks involved in sensing extracellular cues are crucial to all living organisms. Extracellular signals are rapidly transmitted from the cell membrane to the nucleus by activation of enzymatic cascades which ultimately elicit slow changes in gene expression, and thereby affect the cell fate. In the first part of this thesis, the Ras-MAPK cascade transducing signals from the cell membrane to the nucleus is analyzed using mathematical modeling. Model analysis reveals network properties which prevent the MAPK cascade from being inappropriately activated by mutations. Moreover, the simulations unveil a hidden positive feedback loop which ensures strong amplification of MAPK signalling once extracellular stimulation exceeds a certain threshold. The second part of the thesis focuses on how slow gene expression responses feed back into the upstream signalling network. A systematic analysis of gene expression data gathered in mammalian cells demonstrates that such transcriptional feedback generally involves induction of highly unstable signalling inhibitors, thereby establishing negative feedback regulation. Dynamic data-based modelling identifies the SnoN oncoprotein as the central negative feedback regulator in the TGFbeta signalling pathway, and corresponding model predictions are verified experimentally in SnoN-depleted cells. The third part of the thesis focuses on how intracellular signals are decoded by the downstream gene expression machinery. A combined experimental and theoretical analysis of the cyanobacterial iron stress response reveals that small non-coding RNAs allow cells to selectively respond to sufficiently strong and sustained stimuli. Finally, a reverse engineering approach is applied to derive the topology of a complex mammalian transcription factor network from high-throughput knock-down data. In conclusion, this thesis demonstrates how mathematical modelling can support experimental analysis of biological systems.

Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.