Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Targeting UGCG Overcomes Resistance to Lysosomal Autophagy Inhibition.

Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted. SIGNIFICANCE: We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers. This article is highlighted in the In This Issue feature, p. 247.

PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma.

New strategies are needed to enhance the efficacy of anti-programmed cell death protein antibody (anti-PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-ß in macrophages, the latter being a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer.

New plasma and tissue biomarkers of epithelial ovarian cancer (EOC) could improve early diagnosis and post-diagnosis clinical management. Here we investigated tissue staining and tissue secretion of CLIC1 and CLIC4 across EOC subtypes. CLIC1 and CLIC4 are two promising biomarkers we previously showed were elevated in EOC patient sera. Individually, CLIC1 or CLIC4 stained larger percentages of malignant tumors across all EOC subtypes compared with CA125, particularly early stage and mucinous tumors. CLIC4 also stained benign tumors but staining was limited to nuclei; whereas malignant tumors showed diffuse cellular staining of stromal and tumor cells. Both proteins were shed by all EOC subtypes tumors in short term organ culture at more consistent levels than CA125, supporting their potential as pan-subtype serum and tissue biomarkers. Elevated CLIC4 expression, but not CLIC1 expression, was a negative indicator of patient survival, and CLIC4 knockdown in cultured cells decreased cell proliferation and migration indicating a potential role in tumor progression. These results suggest CLIC1 and CLIC4 are promising serum and tissue biomarkers as well as potential therapeutic targets for all EOC subtypes. This justifies development of high throughput serum/plasma biomarker assays to evaluate utility of a biomarker panel consisting of CLIC1, CLIC4 and CA125.

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes.

Anion exchanger 1 (AE1) is a critical transporter and the primary structural scaffold for large macromolecular complexes responsible for erythrocyte membrane flexibility and integrity. We used zero-length crosslinking and mass spectrometry to probe AE1 structures and interactions in intact erythrocyte membranes. An experimentally verified full-length model of AE1 dimers was developed by combining crosslink-defined distance constraints with homology modeling. Previously unresolved cytoplasmic loops in the AE1 C-terminal domain are packed at the domain-domain interface on the cytoplasmic face of the membrane where they anchor the N-terminal domain's location and prevent it from occluding the ion channel. Crosslinks between AE1 dimers and ankyrin-1 indicate the likely topology for AE1 tetramers and suggest that ankyrin-1 wraps around AE1 tetramers, which may stabilize this oligomer state. This interaction and interactions of AE1 with other major erythrocyte membrane proteins show that protein-protein contacts are often substantially more extensive than previously reported.

Probing structures of large protein complexes using zero-length cross-linking.

Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking. Zero-length cross-linking reagents do not add any atoms to the cross-linked species due to the lack of a spacer arm. This provides a major advantage in the form of providing more precise distance constraints as the cross-linkable groups must be within salt bridge distances in order to react. However, identification of cross-linked peptides using these reagents presents unique challenges. We discuss recent efforts by our group to minimize these challenges by using multiple cycles of LC-MS/MS analysis and software specifically developed and optimized for identification of zero-length cross-linked peptides. Representative data utilizing our current protocol are presented and discussed.

Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling.

Peroxiredoxin-6 (PRDX6) is an unusual member of the peroxiredoxin family of antioxidant enzymes that has only one evolutionarily conserved cysteine. It reduces oxidized lipids and reactive oxygen species (ROS) by oxidation of the active-site cysteine (Cys(47)) to a sulfenic acid, but the mechanism for conversion back to a thiol is not completely understood. Moreover, it has phospholipase A2 (PLA2) activity in addition to its peroxidase activity. Interestingly, some biochemical data are inconsistent with a known high-resolution crystal structure of the catalytic intermediate of the protein, and biophysical data indicate that the protein undergoes conformational changes that affect enzyme activity. In order to further elucidate the solution structure of this important enzyme, we used chemical cross-linking coupled with high-resolution MS (CX-MS), with an emphasis on zero-length cross-links. Distance constraints from high confidence cross-links were used in homology modelling experiments to determine a solution structure of the reduced form of the protein. This structure was further evaluated using chemical cross-links produced by several homo-bifunctional amine-reactive cross-linking reagents, which helped to confirm the solution structure. The results show that several regions of the reduced version of human PRDX6 are in a substantially different conformation from that shown for the crystal structure of the peroxidase catalytic intermediate. The differences between these two structures are likely to reflect catalysis-related conformational changes. These studies also demonstrate that CX-MS using zero-length cross-linking is a powerful strategy for probing protein conformational changes that is complementary to alternative methods such as crystallographic, NMR and biophysical studies.

Spectrin tetramer formation is not required for viable development in Drosophila.

The dominant paradigm for spectrin function is that (αß)2-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and ß-spectrins in Drosophila. Wild-type α-spectrin binds to both ß- and ßH-chains with high affinity, resembling other non-erythroid spectrins. However, α-spec(R22S), a tetramerization site mutant homologous to the pathological α-spec(R28S) allele in humans, eliminates detectable binding to ß-spectrin and reduces binding to ßH-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrin(R22S) rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm.

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry.

Conformational changes of macromolecular complexes play key mechanistic roles in many biological processes, but large, highly flexible proteins and protein complexes usually cannot be analyzed by crystallography or NMR. Here, structures and conformational changes of the highly flexible, dynamic red cell spectrin and effects of a common mutation that disrupts red cell membranes were elucidated using chemical cross-linking coupled with mass spectrometry. Interconversion of spectrin between closed dimers, open dimers, and tetramers plays a key role in maintaining red cell shape and membrane integrity, and spectrins in other cell types serve these as well as more diverse functions. Using a minispectrin construct, experimentally verified structures of closed dimers and tetramers were determined by combining distance constraints from zero-length cross-links with molecular models and biophysical data. Subsequent biophysical and structural mass spectrometry characterization of a common hereditary elliptocytosis-related mutation of α-spectrin, L207P, showed that cell membranes were destabilized by a shift of the dimer-tetramer equilibrium toward closed dimers. The structure of αL207P mutant closed dimers provided previously unidentified mechanistic insight into how this mutation, which is located a large distance from the tetramerization site, destabilizes spectrin tetramers and cell membrane integrity.

Enhanced identification of zero-length chemical cross-links using label-free quantitation and high-resolution fragment ion spectra.

Chemical cross-linking coupled to mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. "Zero-length" cross-links have greater value for these applications than longer cross-links because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification. In this study, we combined label-free precursor ion quantitation and targeted tandem mass spectrometry with a new software tool, Zero-length Cross-link Miner (ZXMiner), to form a multitiered analysis strategy. A major, critical objective was to simultaneously achieve very high accuracy with essentially no false-positive cross-link identifications while maintaining a good depth of analysis. Our strategy was optimized on several proteins with known crystal structures. Comparison of ZXMiner to several existing cross-link analysis software showed that other algorithms detected less true positive cross-links and were far less accurate. Although prior use of zero-length cross-linking was typically restricted to small proteins, ZXMiner and the associated strategy enable facile analysis of very large protein complexes. This was demonstrated by identification of zero-length cross-links using purified 526 kDa spectrin heterodimers and intact red cell membranes and membrane skeletons.

The common hereditary elliptocytosis-associated α-spectrin L260P mutation perturbs erythrocyte membranes by stabilizing spectrin in the closed dimer conformation.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are common disorders of erythrocyte shape primarily because of mutations in spectrin. The most common HE/HPP mutations are located distant from the critical αß-spectrin tetramerization site, yet still interfere with formation of spectrin tetramers and destabilize the membrane by unknown mechanisms. To address this question, we studied the common HE-associated mutation, αL260P, in the context of a fully functional mini-spectrin. The mutation exhibited wild-type tetramer binding in univalent binding assays, but reduced binding affinity in bivalent-binding assays. Biophysical analyses demonstrated the mutation-containing domain was only modestly structurally destabilized and helical content was not significantly changed. Gel filtration analysis of the αL260P mini-spectrin indicated more compact structures for dimers and tetramers compared with wild-type. Chemical crosslinking showed structural changes in the mutant mini-spectrin dimer were primarily restricted to the vicinity of the αL260P mutation and indicated large conformational rearrangements of this region. These data indicate the mutation increased the stability of the closed dimer state, thereby reducing tetramer assembly and resulting in membrane destabilization. These results reveal a novel mechanism of erythrocyte membrane destabilization that could contribute to development of therapeutic interventions for mutations in membrane proteins containing spectrin-type domains associated with inherited disease.

A comprehensive model of the spectrin divalent tetramer binding region deduced using homology modeling and chemical cross-linking of a mini-spectrin.

Spectrin dimer-tetramer interconversion is a critical contributor to red cell membrane stability, but some properties of spectrin tetramer formation cannot be studied effectively using monomeric recombinant domains. To address these limitations, a fused αß mini-spectrin was produced that forms wild-type divalent tetramer complexes. Using this mini-spectrin, a medium-resolution structure of a seven-repeat bivalent tetramer was produced using homology modeling coupled with chemical cross-linking. Inter- and intramolecular cross-links provided critical distance constraints for evaluating and optimizing the best conformational model and appropriate docking interfaces. The two strands twist around each other to form a super-coiled, rope-like structure with the AB helix face of one strand associating with the opposing AC helix face. Interestingly, two tetramer site hereditary anemia mutations that exhibit wild-type binding in univalent head-to-head assays are located in the interstrand region. This suggests that perturbations of the interstrand region can destabilize spectrin tetramers and the membrane skeleton. The α subunit N-terminal cross-links to multiple sites on both strands, demonstrating that this non-homologous tail remains flexible and forms heterogeneous structures in the tetramer complex. Although no cross-links were observed involving the ß subunit non-homologous C-terminal tail, several cross-links were observed only when this domain was present, suggesting it induces subtle conformational changes to the tetramer site region. This medium-resolution model provides a basis for further studies of the bivalent spectrin tetramer site, including analysis of functional consequences of interstrand interactions and mutations located at substantial molecular distances from the tetramer site.

Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex.

As the principal component of the membrane skeleton, spectrin confers integrity and flexibility to red cell membranes. Although this network involves many interactions, the most common hemolytic anemia mutations that disrupt erythrocyte morphology affect the spectrin tetramerization domains. Although much is known clinically about the resulting conditions (hereditary elliptocytosis and pyropoikilocytosis), the detailed structural basis for spectrin tetramerization and its disruption by hereditary anemia mutations remains elusive. Thus, to provide further insights into spectrin assembly and tetramer site mutations, a crystal structure of the spectrin tetramerization domain complex has been determined. Architecturally, this complex shows striking resemblance to multirepeat spectrin fragments, with the interacting tetramer site region forming a central, composite repeat. This structure identifies conformational changes in alpha-spectrin that occur upon binding to beta-spectrin, and it reports the first structure of the beta-spectrin tetramerization domain. Analysis of the interaction surfaces indicates an extensive interface dominated by hydrophobic contacts and supplemented by electrostatic complementarity. Analysis of evolutionarily conserved residues suggests additional surfaces that may form important interactions. Finally, mapping of hereditary anemia-related mutations onto the structure demonstrate that most, but not all, local hereditary anemia mutations map to the interacting domains. The potential molecular effects of these mutations are described.

A fused alpha-beta "mini-spectrin" mimics the intact erythrocyte spectrin head-to-head tetramer.

Head-to-head assembly of two spectrin heterodimers to form an actin-cross-linking tetramer is a physiologically dynamic interaction that contributes to red cell membrane integrity. Recombinant beta-spectrin C-terminal and alpha-spectrin N-terminal peptides can form tetramer-like univalent complexes, but they cannot evaluate effects of the open-closed dimer interactions or lateral associations of the two-spectrin strands on tetramer formation. In this study we produced and characterized a fused "mini-spectrin dimer" containing the beta-spectrin C-terminal region linked to the alpha-spectrin N-terminal region. This fused mini-spectrin mimics structural and functional properties of intact, full-length dimers and tetramers, including lateral association of the alpha and beta subunits in the dimer and formation of a closed dimer. High performance liquid chromatography gel filtration analyses of this mini-spectrin provide the first direct non-imaging experimental evidence for open and closed spectrin dimers and show that dimer-tetramer-oligomer interconversion is slow at low temperatures and accelerated at 30 degrees C, analogous to full-length spectrin. This protein exhibits wild type dimer-tetramer dissociation constants of approximately 1 mum at 30 degrees C, independent of initial oligomeric state. Conformational states of the mini-spectrin dimer were probed further using chemical cross-linking, which identified distinct groups of cross-links for "open" and "closed" dimers and confirmed the N-terminal region of alpha-spectrin remains highly flexible in the complex, exhibiting closely analogous structures to those observed for the isolated alpha-spectrin N-terminal using NMR (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844). This fusion protein should serve as a useful template for structural and functional studies of the divalent tetramer site.

The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain.

The KRAB domain is a 75 amino acid transcriptional repression module that is encoded by more than 400 zinc finger protein genes, making it the most abundant repression domain in the human proteome. KRAB-mediated gene silencing requires a direct high affinity interaction with the RBCC domain of KAP-1 co-repressor. The structures of the free KRAB domain or the KRAB-RBCC complex are unknown. To address this, we have performed a systematic biophysical analysis of all KRAB isoforms using purified recombinant proteins. All KRAB domains are predominantly monomeric either alone or in a complex with KAP-1-RBCC protein, while a KRAB-SCAN isoform exists as a stable dimer. The KRAB:KAP-1-RBCC interaction requires only the A box in the context of the KRAB(Ab) or KRAB(AC) but both A and B boxes in the context of KRAB(AB). All isoforms bind the KAP-1-RBCC in a stable, zinc dependent fashion with a stoichiometry of KRAB1:3 RBCC with a zinc content of four atoms per RBCC monomer. Limited proteolysis, mass spectrometry and N-terminal sequence analyses suggest that a core complex comprises the entire RBCC domain of KAP-1 and the AB box of the KRAB domain rendering it resistant to proteolysis. NMR spectroscopy showed that unbound KRAB domain does not exist as a well-folded globular protein in solution but may fold into an ordered structure upon binding to the KAP-1-RBCC protein. This is the first example of a structurally disordered repressor domain that is the most widely conserved silencing domain in tetrapods.