Comparative aspects of polyglutamine binding domain in PQBP-1 among Vertebrata

We investigated the evolutionary conservation of polyglutamine binding protein-1 (PQBP-1) among Vertebrata. PQBP-1s were highly conserved and shared the same domain features including a WW domain, a polar amino acid rich domain (PRD), a nuclear localization signal (NLS), and a C-terminal domain (CTD) among Eutheria, but not always among Vertebrata. PQBP-1s of Vertebrata contained a variable region in the middle portion corresponding to the position of PRD. The full form of PRD including both 7aa and DR/ER repeats was specific to Eutheria. PRD of non-eutherian Amniota was minimal. Amphibia had no PRD. The DR/ER repeat was solo in fishes. Agnatha PRD was also rich in polar amino acids, but contained no repetitive sequence. We investigated 3 polyQ-containing proteins known to interact with PQBP-1: BRN-2, Huntingtin, and ATAXIN-1, and showed a diverse nature of protein-protein interaction in Vertebrata. There appears to be no interaction between PQBP-1 and BRN-2, Huntingtin, or ATAXIN-1 in Amphibia, while the interaction between PQBP-1 and BRN-2 is expected to be conserved among Mammalia, and the interaction between PQBP-1 and Huntingtin or ATAXIN-1 depends on the lineage in Eutheria.     

Quantification of Nuclear Protein Transport using Induced Heterodimerization

Trafficking of proteins between the cytoplasm and nucleus occurs exclusively across the nuclear pore complex of eucaryotic cells. Fundamental aspects of this process affect temporal and spatial parameters, the latter carried out by specific import [nuclear localization sequence (NLS)] and export [nuclear export sequence (NES)] sequences. In this study, we focused on the adaptation of a protein heterodimerization assay to kinetically measure Crm1-mediated nuclear export in living cells using the rapalog AP21967, a heterodimerizing agent and NLS- and NES-containing fusion proteins equipped with distinct AP21967-specific binding motifs. In HeLa cells, we observed rapid nuclear export of the NLS-containing fusion protein in the presence of AP21967, with the extent of this process being a function of the number of AP21967-binding motifs. AP21967-induced nuclear export was specifically inhibited by the Crm1-binding molecule leptomycin B. Half maximal export was achieved after ∼ 10 min. We further applied protein heterodimerization in HeLa cells to study induced NLS-mediated nuclear import. Only in the presence of heterodimerizer AP21967 nuclear import of a cytoplasmically localizing fusion protein was observed. Induced protein heterodimerization is thus a valuable tool to quantitatively study nucleocytoplasmic protein trafficking in cultured cells, in a non-invasive, time-saving manner.     

Interdependent Nucleocytoplasmic Trafficking and Interactions of Dis3 with Rrp6, the Core Exosome and Importin‐α3

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome‐binding protein Dis3, a processive 3′ to 5′ exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N‐terminal domain mutants of dDis3 abolish associations with the ‘core’ exosome, yet only reduce binding to the ‘nuclear’ exosome‐associated factor dRrp6. We show that nuclear localization of dDis3 requires a C‐terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co‐precipitates the NLS‐binding protein importin‐α3. Surprisingly, dDis3 constructs that lack or mutate the C‐terminal NLS retain importin‐α3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin‐α3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.     

Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells

We report phosphorylated and ubiquitinated aggregates of TAR DNA binding protein of 43 kDa (TDP-43) in SH-SY5Y cells similar to those in brains of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). Two candidate sequences for the nuclear localization signal were examined. Deletion of residues 78-84 resulted in cytoplasmic localization of TDP-43, whereas the mutant lacking residues 187-192 localized in nuclei, forming unique dot-like structures. Proteasome inhibition caused these to assemble into phosphorylated and ubiquitinated TDP-43 aggregates. The deletion mutants lacked the exon skipping activity of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Our results suggest that intracellular localization of TDP-43 and proteasomal function may be involved in inclusion formation and neurodegeneration in TDP-43 proteinopathies.     

Redox-Linked Conformational Dynamics in Apoptosis-Inducing Factor

Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH₂-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH.     

A Nuclear Localization Signal at the SAM-SAM Domain Interface of AIDA-1 Suggests a Requirement for Domain Uncoupling Prior to Nuclear Import

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus.     

Structural and Functional Divergence within the Dim1/KsgA Family of rRNA Methyltransferases

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.     

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

The majority of eukaryotic pre-mRNAs are processed by 3′-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3′-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ∼ 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 Å resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3′-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.