Identifikasi Mekanisme Molekuler Senyawa Ftalosianina sebagai Kandidat Photosensitizer pada Terapi Fotodinamika secara In Silico

<p>Bakteri patogen seperti Pseudomonas aeruginosa membutuhkan zat besi untuk dapat mempertahankan kelangsungan hidupnya. HasAp merupakan suatu protein yang dihasilkan oleh bakteri patogen sebagai sumber zat besi tersebut. Protein HasAp selanjutnya akan berikatan dengan protein membran luar yaitu HasR untuk dapat meneruskan sinyal pada sel bakteri. Penyerapan zat besi pada bakteri patogen ini dapat menjadi strategi pengembangan metode terapi dalam mengendalikan dan mencegah penyakit infeksi yang disebabkan oleh bakteri patogen Pseudomonas aeruginosa, salah satunya dengan memanfaatkan ftalosianina sebagai photosensitizer pada terapi fotodinamika. Penelitian ini bertujuan untuk mengidentifikasi, mengevaluasi, dan mengeksplorasi mekanisme aksi senyawa ftalosianina terhadap protein HasAp, serta pengaruhnya pada bagian sisi aktif HasR dengan menggunakan studi <em>in silico</em>. Studi ligan-protein <em>docking </em>dilakukan dengan menggunakan perangkat lunak MGLTools 1.5.6 yang dilengkapi dengan AutoDock 4.2 untuk mengamati afinitas dan interaksi molekuler antara molekul senyawa Fe-ftalosianina (Fe-Pc) dan Ga-ftalosianina (Ga-Pc) terhadap makromolekul protein HasAp. Selanjutnya, studi protein-protein <em>docking</em> dilakukan terhadap sistem kompleks ligan-protein untuk mengamati pengaruhnya terhadap area pengikatan dari makromolekul protein HasR dengan menggunakan perangkat lunak PatchDock. Berdasarkan hasil ligan-protein <em>docking</em>, senyawa Fe-ftalosianina (Fe-Pc) memiliki afinitas paling baik terhadap kedua protein HasAp, yaitu dengan nilai masing-masing -68,45 kJ/mol dan -69,16 kJ/mol. Kemudian, hasil studi protein-protein <em>docking</em> antara kompleks senyawa Fe-ftalosianina (Fe-Pc) dan protein HasAp terhadap protein HasR memiliki nilai <em>Atomic Contact Energy</em> (ACE) positif, yaitu 556,56 kJ/mol. Perbedaan struktur molekul senyawa ftalosianina terbukti mampu mempengaruhi mekanisme aksi terhadap protein target, sehingga hasil studi ini dapat menjadi acuan dalam mendesain struktur senyawa ftalosianina sebagai kandidat photosensitizer dalam terapi fotodinamika.</p><p><span id="docs-internal-guid-a4f8fd6d-7fff-5f66-2ef0-68b87893ec76"><strong><strong>Identification of the Molecular Mechanism of Phthalocyanine Compounds as Photosensitizer Candidates in Photodynamic Therapy by <em>In Silico</em>. </strong></strong>Pathogenic bacteria including <em>Pseudomonas aeruginosa</em> need iron elements to be able to maintain their survival. HasAp is a protein produced by pathogenic bacteria as a source of iron. The HasAp protein will then bind to the outer membrane protein, namely HasR, to be able to forward signals in bacterial cells. Absorption of iron in these pathogenic bacteria can be a strategy for developing therapeutic methods in controlling and preventing infectious diseases caused by pathogenic bacteria <em>Pseudomonas aeruginosa</em>, one of which is by using phthalocyanine as a photosensitizer in photodynamic therapy. This study aims to identify, evaluate, and explore the mechanism of action of phthalocyanine compounds against HasAp proteins, and their effects on the active site of HasR through in silico studies. Ligand-protein docking studies were performed using MGLTools 1.5.6 with AutoDock 4.2 to observe the affinity and molecular interactions between molecules of Fe-phthalocyanine (Fe-Pc) and Ga-phthalocyanine (Ga-Pc) against HasAp protein macromolecules. Furthermore, a protein-protein docking study of the ligand-protein complexes system was simulated to observe its effect on the binding area of the HasR protein macromolecules using PatchDock. Based on the ligand-protein docking results, Fe-phthalocyanine (Fe-Pc) compounds have the best affinity for both HasAp proteins, with a binding energy value of -68.45 kJ/mol and -69.16 kJ/mol, respectively. The protein-protein docking study results between the complex compound Fe-phthalocyanine (Fe-Pc) and HasAp protein against HasR protein have a positive Atomic Contact Energy (ACE) value, with an ACE value of 556.56 kJ/mol. Differences in the molecular structure of phthalocyanine compounds are proven to be able to influence the mechanism of action against the target protein. Therefore, the results of this study can be a reference in designing the structure of phthalocyanine compounds as photosensitizer candidates in photodynamic therapy.</span></p>

Functional Annotation of Hypothetical Proteins Derived from Suppressive Subtraction Hybridization (SSH) Analysis Shows NPR1 (Non-Pathogenesis Related)-Like Activity

Fusarium wilt is considered the most devastating banana disease incited by Fusarium oxysporum f. sp. cubense (FOC). The present study addresses suppressive subtraction hybridization (SSH) analysis for differential gene expression in banana plant, mediated through FOC and its interaction with biocontrol agent Trichoderma asperellum (prr2). SSH analysis yielded a total of 300 clones. The resultant clones were sequenced and processed to obtain 22 contigs and 87 singleton sequences. BLAST2GO (Basic Local Alignment Search Tool 2 Gene Ontology) analysis was performed to assign known protein function. Initial functional annotation showed that contig 21 possesses p38-like endoribonuclease activity and duality in subcellular localization. To gain insights into its additional roles and precise functions, a sequential docking protocol was done to affirm its role in the defense pathway. Atomic contact energies revealed binding affinities in the order of miRNA > phytoalexins > polyubiquitin, emphasizing their role in the Musa defense pathway. Contig 21 and polyubiquitin showed an atomic contact energy value of −479.60 kJ/mol, and even higher atomic contact energies were observed for miRNA (−804.86, −482.28, −494.75 kJ/mol), demonstrating its high RNA-binding properties. Phytoalexin contig 21-interacting interfacial residues were identified as rigid (10)/non-rigid (2) based on Bi, N values, and B-factor per residue. Hence, based on these results, contig 21 was characterized as a NPR1 (non-pathogenesis-related protein) homolog that is involved in plant defense and systemic induced resistance.

Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15

Cadmium (Cd) remediation in Pseudomonas aeruginosa is achieved through the function of two vital genes, cadA and cadR, that code for P-type ATPase (CadA) and transcription regulatory protein (CadR), respectively. Although numerous studies are available on these metal-sensing and regulatory proteins, the promoter of these genes, metal sensing and binding ability, are poorly understood. The present work is aimed at the characterization of the CadR protein, identification of the P cadR promoter and protein–promoter–metal binding affinity using bioinformatics and to validate the results by cloning the P cadR promoter in Escherichia coli DH5α. The promoter regions and its curvature were identified and analysed using PePPER software (University of Groningen, The Netherland) and the Bendit program (Version: v.1.0), respectively. Using Phyre, the three-dimensional structure of CadR was modelled, and the structure was validated by Ramachandran plots. The DNA-binding domain was present in the N-terminal region of CadR. A dimeric interface was observed in helix-turn-helix and metal ion-binding sites at the C-terminal. Docking studies showed higher affinity of Cd to both CadR (Atomic contact energy = −15.04 kcal/Mol) and P cadR (Atomic contact energy = −40.18 kcal/Mol) when compared to other metal ions. CadR with P cadR showed the highest binding affinity (Atomic contact energy= −250.40 kcal/Mol) when compared with P cadA. In vitro studies using green fluorescent protein tagged with P cadR ( gfp-P cadR) cloned in E. coli-expressed gfp protein in a concentration-dependent manner upon Cd exposure. Based on our in silico studies and in vitro molecular cloning analysis, we conclude that P cadR and CadR are active only in the presence of Cd. The CadR protein has the highest binding affinity with P cadR. As it became apparent that the cadR gene regulates the P cadR activity in the presence of Cd with high specificity, and the cadR and P cadR can be used as a biological tool for development of a microbial biosensor.

Evaluation of the energy barrier for failure of Au atomic contact based on temperature dependent current–voltage characteristics

Evaluation of the energy barrier for failure of Au atomic contact based on the current–voltage characteristics as a function of sample temperature.