Parce qu’ils sont la première cause virale d’infections des voies respiratoires supérieures et inférieures, les rhinovirus humains (RVH) constituent un problème majeur de santé publique. À ce jour, aucun vaccin ni antiviral n’est disponible pour lutter contre ces agents pathogènes. Un crible en doublehybride chez la levure nous a permis d’identifier un nouveau partenaire peptidique de la protéase virale à cystéine 2A (2Apro), l’hexapeptide LVLQTM. Ce dernier agit comme un véritable pseudosubstrat de la 2Apro et inhibe son activité. Ce peptide a été modifié chimiquement à son extrémité C-terminale avec un groupement réactif électrophile fluorométhylcétone pour former une liaison covalente avec le groupement thiol nucléophile du site actif de l'enzyme viral. Des expériences réalisées ex vivo et in vivo ont montré que le peptide LVLQTM modifié était un puissant inhibiteur de la réplication du RVH dans les cellules A549 et chez la souris. La structure 3D déjà connue de la 2Apro du RVH-2 a ensuite permis de modéliser la fixation de LVLQTM dans la poche de liaison du substrat de la protéase et la comparaison des séquences des 2Apro des espèces RVH-A, -B et -C a révélé que les résidus impliqués dans l'interaction avec le peptide LVLQTM sont relativement bien conservés. Si le peptide inhibiteur semblait donc agir contre tous les sérotypes de RVH, son utilisation à des fins thérapeutiques pouvait être étendue à d'autres entérovirus puisqu’il inhibait également la 2Apro de l’entérovirus 71 (EV-71) et par conséquent la réplication virale. De plus, la comparaison de la séquence des protéases 2A de l’EV-71 avec celle du RVH-A2 n’a révélé aucune différence majeure. Par conséquent, cette étude ouvre de nouvelles perspectives dans la mise au point d’un antiviral à large spectre d’action contre tous les entérovirus / Human rhinoviruses (HRV) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. To date no vaccine or antiviral are available against these pathogens. Using a high-throughput yeast two-hybrid screening, we identified a six amino acid “hit” peptide, LVLQTM, which acted as a pseudo-substrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified at its C-terminus with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. Based on HRV-2 2Apro crystallographic data, a virtual docking model was then set up to predict the inhibitor binding mode into the ligand binding pocket of the enzyme. Sequence comparison between different 2Apro from HRV-A, -B and –C species revealed that the aminoacid residues involved in the interaction with the inhibitor are relatively well conserved. If our peptide inhibitor seemed to be of general use against all HRV serotypes, its use for therapeutic purposes could be extended to other enterovirus-associated diseases since it was also active against Human Enterovirus 71 (EV-71) 2A proteases and EV-71 replication. Moreover, comparison of the sequence of these proteases with the one of HRV-A2 revealed only minor differences in the residues involved in the interaction with LVLQTM. Therefore, this study opens new doors in the development of an antiviral against a wide range of enteroviruses

Yes / Multidrug resistance (MDR) limits the beneficial outcomes of conventional breast cancer chemotherapy. Ras-related nuclear protein (Ran-GTP) plays a key role in these resistance mechanisms, assisting cancer cells to repair damage to DNA. Herein, we investigate the co-delivery of Ran-RCC1 inhibitory peptide (RAN-IP) and doxorubicin (DOX) to breast cancer cells using liposomal nanocarriers. A liposomal delivery system, co-encapsulating DOX, and RAN-IP, was prepared using a thin-film rehydration technique. Dual-loaded liposomes were optimized by systematic modification of formulation variables. Real-Time-Polymerase Chain Reaction was used to determine Ran-GTP mRNA expression. In vitro cell lines were used to evaluate the effect of loaded liposomes on the viability of breast and lung cancer cell lines. In vivo testing was performed on a murine Solid Ehrlich Carcinoma model. RAN-IP reversed the Ran-expression-mediated MDR by inhibiting the Ran DNA damage repair function. Co-administration of RAN-IP enhanced sensitivity of DOX in breast cancer cell lines. Finally, liposome-mediated co-delivery with RAN-IP improved the anti-tumor effect of DOX in tumor-bearing mice when compared to single therapy. This study is the first to show the simultaneous delivery of RAN-IP and DOX using liposomes can be synergistic with DOX and lead to tumor regression in vitro and in vivo.

A troponina I (TnI) é a sub-unidade inibitória do complexo troponina, responsável pela regulação da contração do músculo esquelético. Foi demonstrado que sua ação inibitória sobre a Mg2+ATPase da actomiosina, deve-se principalmente à região entre os resíduos 96 e 116 (região do peptídeo inibitório). Para estudar o mecanismo de inibição a nível molecular, produzimos três mutantes na região do peptídeo inibitório através de mutações sítio-dirigidas. Substituímos os resíduos lisina 105 por ácido glutâmico (K105E), fenilalanina 106 por tirosina (F106Y) e arginina 113 por ácido glutâmico (R113E). As troponinas I mutantes foram expressas em E.coli, purificadas e ensaiadas em sua atividade inibitória, interações com os outros componentes do complexo regulatório e sua capacidade regulatória. Os resultados obtidos indicam que a mutação na posição 105 alterou a interação da proteína com a tropomiosina, diminuindo sua atividade inibitória e afinidade pela actina-tropomiosina. A substituição na posição 113 alterou a interação da proteína com a actina e com a actina-tropomiosina, também diminuindo a atividade inibitória na presença de tropomiosina e inviabilizando a inibição na ausência de tropomiosina. Já a substituição na posição 106 não produziu alteração detectável. Concluímos que o resíduo 105 faz parte do sítio de ligação da troponina I ao complexo actina-tropomiosina e que o resíduo 113 participa diretamente do mecanismo de inibição. Desta forma, definimos duas interfaces de interação da troponina I com o filamento de actina-tropomiosina, necessárias a ligação da troponina I ao filamento e inibição da ATPase. / Troponin I (TnI) is the inhibitory subunit of the troponin complex, responsible for the regulation of skeletal muscle contraction. It has been demonstrated that TnI\'s inhibitory action on Mg2+ATPase of actomyosin is due principally to the region between residues 96 and 116 (the inhibitory region). To study the inhibitory mechanism at the molecular level, we produced three mutants of the inhibitory region by site-directed mutagenesis. We substituted lysine 105 for glutamic acid (K105E), phenylalanine 106 for tyrosine (F106Y) and arginine 113 for glutamic acid (R113E). The TnI mutants were expressed in E. coli, purified and analyzed for their inhibitory activity, interaction with other components of the regulatory complex and regulatory capacity. The results indicate that the mutation in K105E modified the interaction of TnI with tropomyosin, reduced its inhibitory activity and actin-tropomyosin affinity. The mutant R113E displayed modified interaction with actin and actin-tropomyosin, reduced inhibitory activity in the presence of tropomyosin and essentially no inhibitory activity in the absence of tropomyosin. The mutant F106Y behaved essentially like wild-type TnI. We conclude that residue 105 is part of the site by which troponin I binds to the actin-tropomyosin and that residue 113 participates directly in the inhibitory mechanism. In this way, we have defined two interfaces between troponin I and the actin-tropomyosin which are necessary for binding TnI to the filament and to inhibit the actomyosin ATPase.

Le peptide hCAP18/LL-37, une partie de la défense immunitaire innée, a maintenant été reconnu comme multifonctionnelle pour les cellules eucaryotes. Nos études démontrent sa contribution au développement du cancer, montrant qu'il est surexprimé dans la plupart des tumeurs mammaires humaines, active la signalisation la famille de ERBB et augmente le potentiel métastatique des cellules cancéreuses du sein. Notre comparaison des deux lignées du cancer du sein n'a pas révélé de récepteurs communs, mais une structure peptidique identiques mais de chiralité différente est pré requis pour le peptide dans toutes ses activités. Nous émettons l'hypothèse que LL-37 active indirectement des récepteurs transmembranaires en se liant à la membrane cellulaire. Des peptides tronqués dérivés de LL-37 inhibent ses activités et peuvent aider à concevoir une future thérapie anticancéreuse. / The peptide hCAP18/LL-37, part of the innate immune defense, has now been recognized as multifunctional for eukaryotic cells. Our studies demonstrate its contribution to cancer development, showing that it is overexpressed in most human breast tumors, activates ERBB signaling and increases the metastatic potential of breast cancer cells. Our comparison on two breast cancer lines did not reveal any common receptors but identical structural prerequisites for the peptide in all its activities. We hypothesize that LL-37 indirectly activates transmembrane receptors by attaching to the cellular membrane. Truncated derivatives inhibit its activities and may help to design a future anticancer therapy.

A troponina I (TnI) é a sub-unidade inibitória do complexo troponina, responsável pela regulação da contração do músculo esquelético. Foi demonstrado que sua ação inibitória sobre a Mg2+ATPase da actomiosina, deve-se principalmente à região entre os resíduos 96 e 116 (região do peptídeo inibitório). Para estudar o mecanismo de inibição a nível molecular, produzimos três mutantes na região do peptídeo inibitório através de mutações sítio-dirigidas. Substituímos os resíduos lisina 105 por ácido glutâmico (K105E), fenilalanina 106 por tirosina (F106Y) e arginina 113 por ácido glutâmico (R113E). As troponinas I mutantes foram expressas em E.coli, purificadas e ensaiadas em sua atividade inibitória, interações com os outros componentes do complexo regulatório e sua capacidade regulatória. Os resultados obtidos indicam que a mutação na posição 105 alterou a interação da proteína com a tropomiosina, diminuindo sua atividade inibitória e afinidade pela actina-tropomiosina. A substituição na posição 113 alterou a interação da proteína com a actina e com a actina-tropomiosina, também diminuindo a atividade inibitória na presença de tropomiosina e inviabilizando a inibição na ausência de tropomiosina. Já a substituição na posição 106 não produziu alteração detectável. Concluímos que o resíduo 105 faz parte do sítio de ligação da troponina I ao complexo actina-tropomiosina e que o resíduo 113 participa diretamente do mecanismo de inibição. Desta forma, definimos duas interfaces de interação da troponina I com o filamento de actina-tropomiosina, necessárias a ligação da troponina I ao filamento e inibição da ATPase. / Troponin I (TnI) is the inhibitory subunit of the troponin complex, responsible for the regulation of skeletal muscle contraction. It has been demonstrated that TnI\'s inhibitory action on Mg2+ATPase of actomyosin is due principally to the region between residues 96 and 116 (the inhibitory region). To study the inhibitory mechanism at the molecular level, we produced three mutants of the inhibitory region by site-directed mutagenesis. We substituted lysine 105 for glutamic acid (K105E), phenylalanine 106 for tyrosine (F106Y) and arginine 113 for glutamic acid (R113E). The TnI mutants were expressed in E. coli, purified and analyzed for their inhibitory activity, interaction with other components of the regulatory complex and regulatory capacity. The results indicate that the mutation in K105E modified the interaction of TnI with tropomyosin, reduced its inhibitory activity and actin-tropomyosin affinity. The mutant R113E displayed modified interaction with actin and actin-tropomyosin, reduced inhibitory activity in the presence of tropomyosin and essentially no inhibitory activity in the absence of tropomyosin. The mutant F106Y behaved essentially like wild-type TnI. We conclude that residue 105 is part of the site by which troponin I binds to the actin-tropomyosin and that residue 113 participates directly in the inhibitory mechanism. In this way, we have defined two interfaces between troponin I and the actin-tropomyosin which are necessary for binding TnI to the filament and to inhibit the actomyosin ATPase.

Indiana University-Purdue University Indianapolis (IUPUI) / Computational protein design aims at designing amino acid sequences that can fold into a target structure and perform a desired function. Many computational design methods have been developed and their applications have been successful during past two decades. However, the success rate of protein design remains too low to be of a useful tool by biochemists whom are not an expert of computational biology. In this dissertation, we first developed novel computational assessment techniques to assess several state-of-the-art computational techniques. We found that significant progresses were made in several important measures by two new scoring functions from RosettaDesign and from OSCAR-design, respectively. We also developed the first machine-learning technique called SPIN that predicts a sequence profile compatible to a given structure with a novel nonlocal energy-based feature. The accuracy of predicted sequences is comparable to RosettaDesign in term of sequence identity to wild type sequences. In the last two application chapters, we have designed self-inhibitory peptides of Escherichia coli methionine aminopeptidase (EcMetAP) and de novo designed barstar. Several peptides were confirmed inhibition of EcMetAP at the micromole-range 50% inhibitory concentration. Meanwhile, the assessment of designed barstar sequences indicates the improvement of OSCAR-design over RosettaDesign.