Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

No description available.

No description available.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

No description available.

Lentiviruses can infect non-dividing cells, and various cellular nuclear transport proteins provide crucial functions for lentiviral nuclear entry and integration. Genome-wide small interfering RNA screens previously identified nuclear pore complex component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). We found that HIV-1 infection of NUP153 depleted cells resulted in normal levels of reverse transcription, a moderate reduction of 2-long terminal repeat circles, and a relatively large reduction in integrated proviruses, consistent with a role for NUP153 during nuclear entry of the HIV-1 pre-integration complex. We ascertained the capsid (CA) to be the major viral determinant for NUP153 dependence during infection, and accordingly observed a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153C). NUP153C fused to the effector domains of the rhesus Trim5alpha restriction factor (Trim-NUP153C) potently restricted HIV-1, providing an intracellular readout for the NUP153C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153C under these conditions, results that correlated with direct binding between purified recombinant proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 function during infection. Mutagenesis experiments identified NUP153C and CA residues important for binding, and different FG motifs within NUP153C mediated binding to HIV-1 versus EIAV CA proteins. HIV-1 CA binding mapped to residues that line a common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 each competed with NUP153C for binding to HIV-1 CA, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicated that the NUP153C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. We propose that HIV-1 CA binds NUP153 FG motifs to affect viral nuclear import, serving as a novel example of viral hijacking of a fundamental cellular process.

No description available.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.