Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway.

Membrane proteins constitute a significant fraction of the proteome and are important drug targets. While the transmembrane (TM) segments of these proteins are primarily composed of hydrophobic residues, the inclusion of polar residues-either naturally occurring or as a consequence of a disease-related mutation-places a significant folding burden in this environment, potentially impacting bilayer insertion and/or association of neighboring TM helices. Here we investigate the role of an anionic detergent, sodium dodecylsulfate (SDS), and a zwitterionic detergent, dodecylphosphocholine (DPC), in the folding process, and the effects induced by a single polar substitution, on structure and topology of model α-helical TM segments. The peptides, represented by KK-YAAAIAAIAWAXAAIAAAIAA-KKK-NH(2), where X is I or N, are designed with high aqueous solubilities, through poly-lysine tags. Circular dichroism (CD) and NMR were used to monitor peptide secondary structure and diffusional mobility of both peptide and the detergent hosts. For both peptides, SDS binding commenced at a concentration below its CMC, due to Coulombic attraction of anionic SDS to cationic Lys residues. Increasing SDS binding correlated with increasing peptide helicity. Pulsed field gradient (PFG) NMR diffusion measurements revealed that the Asn-containing peptide bound four fewer detergent molecules, corresponding to ca. 20% less SDS than bound by the Ile peptide. Conversely, zwitterionic DPC binding to either peptide was not observed until the DPC concentration approached its CMC. Our findings confirm quantitatively that a single polar residue within a TM segment may have a significant influence on its local membrane environment.

Cellular uptake of substrate-initiated cell-penetrating poly(disulfide)s.

Substrate-initiated, self-inactivating, cell-penetrating poly(disulfide)s (siCPDs) are introduced as general transporters for the covalent delivery of unmodified substrates of free choice. With ring-opening disulfide-exchange polymerization, we show that guanidinium-rich siCPDs grow on fluorescent substrates within minutes under the mildest conditions. The most active siCPD transporters reach the cytosol of HeLa cells within 5 min and depolymerize in less than 1 min to release the native substrate. Depolymerized right after use, the best siCPDs are nontoxic under conditions where cell-penetrating peptides (CPPs) are cytotoxic. Intracellular localization (cytosol, nucleoli, endosomes) is independent of the substrate and can be varied on demand, through choice of polymer composition. Insensitivity to endocytosis inhibitors and classical structural variations (hydrophobicity, aromaticity, branching, boronic acids) suggest that the best siCPDs act differently. Supported by experimental evidence, a unique combination of the counterion-mediated translocation of CPPs with the underexplored, thiol-mediated covalent translocation is considered to account for this decisive difference.

Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms.

High-resolution structural analysis of membrane proteins by X-ray crystallography or solution NMR spectroscopy often requires their solubilization in the membrane-mimetic environments of detergents. Yet the choice of a detergent suitable for a given study remains largely empirical. In the present work, we considered the micelle-crystallized structures of lactose permease (LacY), the sodium/galactose symporter (vSGLT), the vitamin B(12) transporter (BtuCD), and the arginine/agmatine antiporter (AdiC). Representative transmembrane (TM) segments were selected from these proteins based on their relative contact(s) with water, lipid, and/or within the protein, and were synthesized as Lys-tagged peptides. Each peptide was studied by circular dichroism and fluorescence spectroscopy in water, and in the presence of the detergents sodium dodecylsulfate (SDS, anionic); n-dodecyl phosphatidylcholine (DPC, zwitterionic); n-dodecyl-ß-d-maltoside (DDM, neutral); and n-octyl-ß-d-glucoside (OG, neutral, varying acyl tail length). We found that (i) the secondary structures of the TM segments were statistically indistinguishable in the four detergents studied; and (ii) a strong correlation exists between the extent of helical structure of each individual TM segment in detergents with its helicity level as it exists in the full-length protein, indicating that helix adoption is fundamentally the same in both environments. The denaturing properties of so-called 'harsh' detergents may thus largely be due to their interactions with non-membranous regions of proteins. Given the consistency of structural features observed for each TM segment in a variety of micellar media, the overall results suggest that the structure likely corresponds to its relevant biological form in the intact protein in its native lipid bilayer environment.

Positions of polar amino acids alter interactions between transmembrane segments and detergents.

α-Helical transmembrane (TM) segments in membrane proteins are comprised primarily of hydrophobic amino acids that accommodate insertion from water into the nonpolar membrane bilayer. In many such segments, however, polar residues are also present for structural or functional reasons. These latter residues impair the local favorable acyl interactions required for solvation by hydrophobic media such as phospholipids in native bilayers or detergents used for in vitro characterization. Using a series of Lys-tagged designed TM-like peptides (typified by KK-YAAAIAAIAWAIAAIAAAIAA-KKK) in which single-Asn residue substitutions (from Ile or Ala) were made successively from the center of the hydrophobic region toward the C-terminus, we demonstrate that polar residues strongly alter the nature of the interaction between TM segments and the solvating detergent. Through the application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, and tryptophan fluorescence, we observed drastic differences in the structures of the detergent-peptide complexes that contain relatively minor sequence differences. For example, the blue shift of the Trp fluorescence (indicating local detergent solvation at this location) differs by as much as ~10 nm depending upon the position of a single Asn substitution in an otherwise identical segment. The overall results suggest that polar point mutations occurring in a biological membrane will elicit comparable effects, placing a significant refolding burden on the local protein structure and potentially leading to disease states through altered protein--lipid interactions in membrane proteins.

Peptide models of membrane protein folding.

Given the central roles of membrane proteins in cellular processes ranging from nutrient uptake to cell-cell communication, as well as the importance of these proteins as drug targets, efforts to understand and control their structures are vital in human health and disease. The rational design of membrane proteins with modified properties is thus a highly desirable goal in molecular medicine and biotechnology. However, experimental data showing how individual transmembrane (TM) residues and/or segments direct the packing and folding of membrane proteins into biologically functional entities remain sparse. To address these questions in a systematic manner, helix-helix interactions between two (or more) TM segments must be identified and analyzed. Here we present an overview of the utilization of peptides as models of the TM segments of alpha-helical membrane proteins in uncovering the amino acid sequence motifs and interactions that build these molecules. TM peptide design and production strategies are discussed, and specific examples of the application of TM peptides to the study of membrane proteins are presented. We demonstrate that TM peptides can be routinely produced in sufficient quantities for biophysical analysis, are amenable to a variety of experimental techniques, and can effectively replicate the native helix-helix contacts and key aspects of the natural biological structures of membrane proteins.

SDS micelles as a membrane-mimetic environment for transmembrane segments.

An inherent dilemma in the study of the structural biology of membrane proteins is that it is often necessary to use detergents to mimic the native lipid bilayer environment. This situation is of particular interest because the generation of high-resolution structures (through X-ray crystallography and solution NMR) has overwhelmingly relied upon identification of detergents in which membrane proteins may be solubilized without denaturation into a nonbiological state. While sodium dodecyl sulfate (SDS) is perhaps the most widely employed micelle-forming detergent for laboratory procedures involving membrane proteins, it has generally been regarded as a "harsh" detergent synonymous with membrane protein denaturation. Here we investigate systematically the SDS-solubilized states of a series of model alpha-helical transmembrane (TM) segments of varying Ala and Ile content in conjunction with selected single-Asn polar substitutions. Using Lys-tagged peptides typified by KKKKK-FAIAIAIIAWAIAIIAIAIAI-KKKKK in a series of circular dichroism, fluorescence, TOXCAT dimerization assay, and SDS-PAGE migration experiments, we find that both the local environment of the individual peptide helical surfaces and the formation of oligomeric states within the SDS-peptide complex are highly sensitive to point changes in peptide sequence, particularly with respect to local segment hydrophobicity and polar residue position. The overall results suggest that detergent micelles formed from SDS are largely capable of mimicking the tertiary interactions of protein-, lipid-, and aqueous-exposed helical surfaces that arise in the folded TM domains of proteins. The molecular characteristics of SDS-peptide complexes may thus portend a corresponding role for similar TM sequences in the in vivo assembly of polytopic membrane proteins.

Mitochondrial Targeting of Doxorubicin Eliminates Nuclear Effects Associated with Cardiotoxicity.

The highly effective anticancer agent doxorubicin (Dox) is a frontline drug used to treat a number of cancers. While Dox has a high level of activity against cancer cells, its clinical use is often complicated by dose-limiting cardiotoxicity. While this side effect has been linked to the drug's direct activity in the mitochondria of cardiac cells, recent studies have shown that these result primarily from downstream effects of nuclear DNA damage. Our lab has developed a mitochondrially targeted derivative of Dox that enables the selective study of toxicity generated by the presence of Dox in the mitochondria of human cells. We demonstrate that mitochondria-targeted doxorubicin (mtDox) lacks any direct nuclear effects in H9c2 rat cardiomyocytes, and that these cells are able to undergo mitochondrial biogenesis. This recovery response compensates for the mitotoxic effects of Dox and prevents cell death in cardiomyocytes. Furthermore, cardiac toxicity was only observed in Dox but not mtDox treated mice. This study supports the hypothesis that mitochondrial damage is not the main source of the cardiotoxic effects of Dox.

Molecular vehicles for mitochondrial chemical biology and drug delivery.

The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged. Access to mitochondria-specific delivery vectors has allowed the study of biological processes within this intracellular compartment with a heightened level of specificity. In this review, we summarize the features of existing delivery vectors developed for targeting probes and therapeutics to this highly impermeable organelle. We also discuss the major applications of mitochondrial targeting of bioactive molecules, which include the detection and treatment of oxidative damage, combating bacterial infections, and the development of new therapeutic approaches for cancer. Future directions include the assessment of the therapeutic benefit achieved by mitochondrial targeting for treatment of disease in vivo. In addition, the availability of mitochondria-specific chemical probes will allow the elucidation of the details of biological processes that occur within this cellular compartment.

Targeted delivery of doxorubicin to mitochondria.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they disrupt nucleic acid synthesis in the nucleus. Much less is known, however, about whether interfering with nucleic acid synthesis in the mitochondria would have significant cellular effects. In this study, we explore this with a mitochondrially targeted form of the anticancer drug doxorubicin, which inhibits DNA topoisomerase II, an enzyme that is both in mitochondria and nuclei of human cells. When doxorubicin is attached to a peptide that targets mitochondria, it exhibits significant toxicity. However, when challenged with a cell line that overexpresses a common efflux pump, it does not exhibit the reduced activity of the nuclear-localized parent drug and resists being removed from the cell. These results indicate that targeting drugs to the mitochondria provides a means to limit drug efflux and provide evidence that a mitochondrially targeted DNA topoisomerase poison is active within the organelle.

Design, expression, and purification of de novo transmembrane "hairpin" peptides.

While our understanding of the folding and structure of water-soluble proteins has progressed to the point where they can be artificially designed and produced from first principles, there has been only limited work toward the de novo design of membrane proteins. Such studies have been hindered in large part due to the practical challenges in the production and characterization of multispanning transmembrane (TM) proteins that arise from their highly hydrophobic character. In this work, we used molecular biology cloning techniques to produce a library of partially randomized Ala- and Ile-rich de novo helix-loop-helix (hairpin) TM constructs as models for tertiary TM-TM folding. From this plasmid DNA library, we selected sequences corresponding to hairpins with 0, 1, or 2 putative TM segments. While purification protocols could be adapted for application with a broad range of designed protein hairpins, bacterial expression of constructs with multiple predicted TM segments was limited as it is with native membrane proteins. Examples of the peptide hairpins obtained were characterized by circular dichroism spectroscopy, tryptophan fluorescence, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that hairpins composed of two TM segments display characteristic behavior on detergent solubilization, such as an increase in helical structure (vs. that in aqueous buffer), and sequence-dependent migration rates in SDS-PAGE analysis-features that may serve as structural hallmarks to verify dual TM topology in hairpin sequences.