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Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xvii, 250 p.; also includes graphics (some col.) Includes bibliographical references (p. 207-250). Available online via OhioLINK's ETD Center

Memoria para optar al título profesional de Bioquímico / El virus HTLV-I (Virus Linfotrópico humano Tipo I) es el agente etiológico de dos patologías principales, la Mielopatía asociada al HTLV-I/Paraparesia Espástica Tropical (HAM/TSP), una axonopatía neurodegenerativa, donde existe falla en el transporte axonal y la Leucemia de células T del adulto (ATL). No se ha detectado infección por HTLV-I en neuronas, desconociéndose el mecanismo por medio del cual se produce la neurodegeneración en HAM/TSP. Puesto que el reservorio principal del virus son los linfocitos T CD4+, algunos productos virales secretados por estos linfocitos infectados infiltrados en el sistema nervioso central, podrían afectar vías de transducción de señales relacionadas con la reorganización de citoesqueletos y transporte axonal, produciendo el daño axonal de distal a proximal observado en pacientes HAM/TSP. Los productos secretados por células MT2 (linfocitos infectados con HTLV-I) provocan retracción en células de neuroblastoma SH-SY5Y diferenciadas y disminuyen la velocidad de crecimiento neurítico en células PC12 durante su diferenciación a tipo neuronal. Entre las proteínas virales secretadas, se encuentra Tax, la disminución de la velocidad de crecimiento neurítico en células PC12, podría estar mediada por esta proteína viral a través de la vía transduccional que involucra a semaforinas y sus receptores Plexinas. Como un ejemplo, en el caso de Sema3A-Plexin A, un aumento de la actividad de Cdk5 causa hiperfosforilación de Tau y CRMP-2. El fallo en el transporte axoplásmico, podría explicarse por una desregulación de la fosforilación de las subunidades de los motores moleculares kinesina KIF5 y dineína citoplasmática Dync en este escenario. En este trabajo, se propuso estudiar el efecto de los productos secretados desde linfocitos infectados que incluyen Tax sobre el proceso de diferenciación neuronal. Para esto, se utilizó la línea celular de feocromocitoma de rata, células PC12 diferenciables a tipo neuronal por acción de NGF. La Hipótesis planteada es la siguiente: “La exposición a productos secretados de linfocitos infectados por HTLV-I provoca un aumento en el estado de fosforilación de proteínas motoras en modelo neuronal de células PC12” El primer objetivo desarrollado, fue determinar si Tax es uno de los productos virales secretados desde medio de cultivo de células MT-2 responsable del efecto de retardo de crecimiento neurítico de PC12. La adición al cultivo celular de anticuerpos monoclonales anti-Tax mostró que se bloqueó este efecto de retardo de crecimiento, lo que apunta a que este efecto se debe a la acción de Tax presente en el medio de secreción de las células MT-2. El segundo objetivo, contempló estandarizar protocolos de inmunodetección selectivos para determinar las subunidades asociadas al control de funcionalidad de kinesina KIF5 y dineína citoplasmática en células PC12. Los análisis por Western blot mostraron que las cantidades de las subunidades de kinesina KIF5 (cadena pesada y liviana) y dineína citoplasmática (cadena intermedia) presentes en las células PC12, no varían durante el período de diferenciación estudiado. Esto significa, que Tax no estaría alterando su expresión proteica. El tercer objetivo planteado fue comparar el grado de fosforilación de las subunidades de proteínas motoras en células expuestas a productos secretados desde células MT-2 con la condición control con medio de cultivo de la línea celular K562. Al no disponer de anticuerpos contra las formas fosforiladas de estas proteínas motoras, se procedió a separarlas mediante la técnica de electroforesis de geles en dos dimensiones. Este objetivo requirió inicialmente estandarizar esta técnica y luego se emplearon las muestras de lisados de PC12 provenientes del tercer día de diferenciación que habían sido sometidas al medio condicionado MT-2 y al de K562. Estos análisis no mostraron diferencias significativas en las migraciones electroforéticas, que podrían dar cuenta de cambios del estado de fosforilación de estas subunidades motoras. No obstante, estos resultados no se puede descartar que existan diferencias en el grado de fosforilación de las subunidades de estos motores moleculares, puesto que las diferencias en el grado de fosforilación pueden ser muy menores para ser observadas como cambios en la migración electroforética. Se debiera usar un método inmunológico más sensible empleando anticuerpos que reconozcan fosforilaciones determinadas estas proteínas / Effect of secretable proteins from HTLV-I infected lymphocytes on phosphorylation of motor proteins in PC12 cells as a model of neuronal culture HTLV-I virus (Human T-cell lymphotropic virus type I) is the etiologic agent of two main pathologies, the Myelopathy associated to HTLV-I/Tropical Spastic Paraparesis (HAM/TSP), a neurodegenerative axonopathy with impairment of axonal transport, and the Leukemia of adult T-cells (ATL). No infection in neurons has been detected, being unknown the mechanisms of the neurodegeneration in HAM/TSP. Lymphocytes T-CD4+ represent the main reservoir of the virus. Therefore, some secreted products from these infected lymphocytes infiltrated into the central nervous system could produce alteration of signal transduction pathways related with cytoskeletons and axonal transport, thus accounting for the distal to proximal axonal damage observed in HAM/TSP patients. Secreted products from MT-2 cells (HTLV-I infected lymphopcytes cell line) produce retraction in differentiated neuroblastoma SH-SY5Y cells and reduced the rate of neurite growing during PC12 cell differentiation to neuronal type. Among the viral secretable proteins is Tax, hence, the reduction of PC12 neurite growing could be mediated by this viral protein through transduction signalings that involves semaphorins and their receptor Plexins. As an example, in the case of Sema3A-Plexin A, an increase in Cdk5 activity causes Tau and CRMP-2 hyperphoshorylations. The failure of axonal transport could be explained by deregulation of phosphorylation of molecular motor subunits kinesin KIF5 and cytoplasmatic dynein Dync. In this work it was proposed to study the effect of secreted products, including Tax, from infected lymphocytes on the neuronal differentiation process. We employed the rat pheochromocytoma cell line, PC12 cells during NGF-differentiation to neuronal type. The Hypothesis stated expresses that: “Exposure to secreted products from HTLV-I infected lymphocytes causes an increase in the phosphorylation state of motor proteins in a neuronal model of PC12 cells” The first goal developed was to determine if Tax was one of the viral secreted products to the culture medium of MT-2 cell line responsible of retardation of PC12 neurite growing. Addition to the cell culture monoclonal antibodies anti-Tax blocked the effect on retardation rate of neurite growing, this pointed to the action of Tax present in the secretion medium of MT-2 cells. The second goal consisted in the standardization of selective protocols for immunodetection of subunits associated with the functional control of kinesin KIF5 and cytoplasmic dynein present in PC12 cells. Western blots analyses showed that levels of kinesin KiF5 (heavy and light chains) and cytoplasmic dynein (intermediate chain) present in PC12 cells did not change during the followed period of differentiation. This result suggests that their protein expression is not altered by Tax. The third goal included the comparison of phosphorylation levels of the subunits of the motor proteins exposed to MT-2 cell secreted products with the control condition with K562 cell line medium. Due to non-availability of the antibodies against phosphorylated forms of motor proteins, we separated them by two-dimensional gel electrophoresis. This goal initially required the standardization of the technique, and then PC12 lysates obtained at third day of differentiation in the presence of either MT-2 or K562 cell culture medium were used. These analyses did not show significant differences in the electrophoresis migrations of KiF5 and dynein that could account for changes in the phosphorylation levels of these subunits of motor proteins. In spite of our results, it cannot be discarded the existence of differences in the phosphorylation degree of the molecular motor subunits because the differences in the phosphorylation degree could be minors and undistinguishable by electrophoretic migration changes. Thereby, it should be used more sensitive immunological methods employing antibodies capable of distinguish motor proteins phosphorylations

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax is the major viral transactivator and transforming protein centrally involved in the proviral transcription, transformation and proliferation of infected T-cells as well as progression of diseases caused by HTLV-1 infection. Salt-inducible kinases (SIKs) are serine/threonine protein kinases belonging to the AMPK-related kinase (AMPK-RK) family. SIK subfamily consists of three isoforms named SIK1, SIK2 and SIK3 respectively. We have previously demonstrated the negative regulatory role of SIK1 in Tax-mediated activation of proviral transcription from long terminal repeats (LTR). In this study, we reported that both SIK2 and SIK3 exhibited a kinase-dependent suppressive effect on Tax-activated LTR transcription. We also found that SIK1, SIK2 and SIK3 act additively to suppress Tax activation of LTR. We further demonstrated that the SIK2- and SIK3-mediated suppression on LTR transcription was achieved through phosphorylation of TORC1, an essential transcriptional coactivator of CREB required for Tax-mediated transcriptional activation of LTR. Our findings revealed a new function of SIK2 and SIK3 in host restriction to HTLV-1 transcription. Pharmaceutical activation of SIKs or upstream kinase such as LKB1 may provide a new strategy for anti-HTLV-1 therapy. / published_or_final_version / Biochemistry / Master / Master of Medical Sciences

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Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 230 p.; also includes graphics. Includes bibliographical references (p. 200-230). Available online via OhioLINK's ETD Center

No description available.

This thesis presents studies on the interaction between the human T cell lymphotropic virus type 1 (HTLV-1) Tax protein and the 20S proteasome and the role of the interaction in cellular processes and the cytotoxic T cell (CTL) response against HTLV-1. The rapid translocation of Tax into the nucleus is described. Tax accumulates in the nucleus and forms unique bodies involved in transcriptional activation. It was further found that Tax associated with assembled nuclear 20S proteasomes and stimulated the chymotryptic and tryptic activities of the 20S proteasome, independent of the induction of the LMP2 and LMP7 proteasome subunits. Confocal microscopy revealed a partial colocalisation of Tax with nuclear proteasomes. A panel of Tax mutants was generated and their subcellular localisation and association with the 20S proteasome analysed. This analysis revealed that both the N- and C-terminus of Tax play a role in proteasome binding of Tax and further showed that proteasome binding was not sufficient for nuclear localisation of Tax. Therefore, Tax probably translocates into the nucleus prior to and independent of proteasome association. Tax specific CTL clones were generated and characterised using tetrameric MHC class I/peptide complexes. These CTL clones were used to investigate the requirements for processing and presentation of Tax for recognition by CTL. It was found that Tax was a metabolically very stable protein and that the presentation of the immunodominant Tax 11-19 epitope was dependent on the transporter associated with antigen presentation (TAP), independent of the expression of LMP2 and LMP7 proteasome subunits and resistant to treatment with the proteasome inhibitor lactacystin. It is proposed that the interaction between Tax and the 20S proteasome plays a role in Tax mediated transcriptional activation, leading to cellular activation and proliferation, and may not determine the immunodominance of Tax in the CTL response against HTLV-1.