ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes.

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Translocation of a Cell Surface Spliceosomal Complex Induces Alternative Splicing Events and Lymphoma Cell Necrosis.

Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.

Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach.

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~1011 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 106 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.

SQID-XLink: implementation of an intensity-incorporated algorithm for cross-linked peptide identification.

SUMMARY: Peptide identification algorithm is a major bottleneck for mass spectrometry based chemical cross-linking experiments. Our lab recently developed an intensity-incorporated peptide identification algorithm, and here we implemented this scheme for cross-linked peptide discovery. Our program, SQID-XLink, searches all regular, dead-end, intra and inter cross-linked peptides simultaneously, and its effectiveness is validated by testing a published dataset. This new algorithm provides an alternative approach for high confidence cross-linking identification. AVAILABILITY: SQID-XLink program is freely available for download from http://quiz2.chem.arizona.edu/wysocki/bioinformatics.htm SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: vwysocki@email.arizona.edu.

Assembly of human frataxin is a mechanism for detoxifying redox-active iron.

Mitochondrial function depends on a continuous supply of iron to the iron-sulfur cluster (ISC) and heme biosynthetic pathways as well as on the ability to prevent iron-catalyzed oxidative damage. The mitochondrial protein frataxin plays a key role in these processes by a novel mechanism that remains to be fully elucidated. Recombinant yeast and human frataxin are able to self-associate in large molecular assemblies that bind and store iron as a ferrihydrite mineral. Moreover, either single monomers or polymers of human frataxin have been shown to serve as donors of Fe(II) to ISC scaffold proteins, oxidatively inactivated [3Fe-4S](+) aconitase, and ferrochelatase. These results suggest that frataxin can use different molecular forms to accomplish its functions. Here, stable monomeric and assembled forms of human frataxin purified from Escherichia coli have provided a tool for testing this hypothesis at the biochemical level. We show that human frataxin can enhance the availability of Fe(II) in monomeric or assembled form. However, the monomer is unable to prevent iron-catalyzed radical reactions and the formation of insoluble ferric iron oxides. In contrast, the assembled protein has ferroxidase activity and detoxifies redox-active iron by sequestering it in a protein-protected compartment.

Supramolecular assemblies of human frataxin are formed via subunit-subunit interactions mediated by a non-conserved amino-terminal region.

The mitochondrial protein frataxin is emerging as a novel mechanism to promote iron metabolism while also providing anti-oxidant protection. Recombinant frataxin proteins from different species are able to form large molecular assemblies that store Fe(III) as a stable mineral in vitro. Furthermore, monomeric and assembled forms of frataxin donate Fe(II) to the Fe-S cluster scaffold protein IscU, [3Fe-4S]1+ aconitase, and ferrochelatase in vitro. However, little is known about the speciation of frataxin in vivo, and the physiologically relevant form(s) of the protein remains undefined. Here, we report that human heart mitochondria contain frataxin species of increasing negative surface charge and molecular mass, ranging from monomer to polymers of >1 MDa. Moreover, we show that the main partner protein of frataxin, IscU, binds in a stable manner to frataxin oligomers. These results suggest that assembly is a physiologic property of frataxin. Biochemical analyses further reveal that, unlike the prokaryotic and yeast frataxin homologues, which require iron-protein interactions for assembly, human frataxin uses stable subunit-subunit interactions involving a non-conserved amino-terminal region. We propose that human frataxin is a modular protein that depends on self-assembly to accomplish its diverse functions.

The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast.

Mitochondrial ferritin (MtF) is structurally and functionally similar to the cytosolic ferritins, molecules designed to store and detoxify cellular iron. MtF expression in human and mouse is restricted to the testis and few tissues, and it is abundant in the erythroblasts of patients with sideroblastic anemia, where it is thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast mitochondria and processed to functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the respiratory deficiency caused by the loss of frataxin protecting the activity of iron-sulfur enzymes and enabling frataxin-deficient cells to grow on non-fermentable carbon sources. Furthermore, MtF expression prevented the development of mitochondrial iron overload, preserved mitochondrial DNA integrity and increased cell resistance to H2O2. The data show that MtF can substitute for most frataxin functions in yeast, suggesting that frataxin is directly involved in mitochondrial iron-binding and detoxification.

Frataxin acts as an iron chaperone protein to modulate mitochondrial aconitase activity.

Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity. Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase. We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants. Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive [3Fe-4S]1+ enzyme to the active [4Fe-4S]2+ form of the protein. Thus, frataxin is an iron chaperone protein that protects the aconitase [4Fe-4S]2+ cluster from disassembly and promotes enzyme reactivation.

Structure of frataxin iron cores: an X-ray absorption spectroscopic study.

X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.

Yeast frataxin sequentially chaperones and stores iron by coupling protein assembly with iron oxidation.

We have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters >2000 atoms of iron in 2-4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --> alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with accumulation of 50-75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store.

Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of frataxin, a conserved mitochondrial protein of unknown function. Mitochondrial iron accumulation, loss of iron-sulfur cluster-containing enzymes and increased oxidative damage occur in yeast and mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA patients, suggesting that frataxin may be involved in export of iron from the mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative damage. We have previously shown that yeast frataxin has structural and functional features of an iron storage protein. In this study we have investigated the function of human frataxin in Escherichia coli and Saccharomyces cerevisiae. When expressed in E.coli, the mature form of human frataxin assembles into a stable homopolymer that can bind approximately 10 atoms of iron per molecule of frataxin. The iron-loaded homopolymer can be detected on non-denaturing gels by either protein or iron staining demonstrating a stable association between frataxin and iron. As analyzed by gel filtration and electron microscopy, the homopolymer consists of globular particles of approximately 1 MDa and ordered rod-shaped polymers of these particles that accumulate small electron-dense cores. When the human frataxin precursor is expressed in S.cerevisiae, the mitochondrially generated mature form is separated by gel filtration into monomer and a high molecular weight pool of >600 kDa. A high molecular weight pool of frataxin is also present in mouse heart indicating that frataxin can assemble under native conditions. In radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation with approximately five atoms of (55)Fe bound per molecule. These findings suggest that FRDA results from decreased mitochondrial iron storage due to frataxin deficiency which may impair iron metabolism, promote oxidative damage and lead to progressive iron accumulation.