Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.     

Resequencing the Mycobacterium avium subsp. paratuberculosis K10 Genome: Improved Annotation and Revised Genome Sequence

We report the resequencing and revised annotation of the Mycobacterium avium subsp. paratuberculosis K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.The American bovine isolate K10 remains the only Mycobacterium avium subsp. paratuberculosis genome to be fully sequenced and published to date (1). Although this 4.8-Mbp genome likely contains some assembly errors (3), it has provided, and will continue to provide, an invaluable resource for Mycobacterium research. The assembly errors were identified through optical mapping of related M. avium subsp. paratuberculosis strain ATCC 19698, which revealed a 648-kb inversion around the origin of replication and two additional copies of the insertion sequences IS1311 and IS_MAP03 (3). These findings were subsequently validated via PCR, Southern blotting, and (Sanger) sequence analysis in ATCC 19698 and were also confirmed to be present in K10 (3). We designate this interim corrected genome M. avium subsp. paratuberculosis K10′. To further improve this resource, we undertook a resequencing project of the original M. avium subsp. paratuberculosis K10 genome.Whole-genome sequencing was performed on the Illumina GAIIx platform using one flow cell lane with 36-cycle paired-end chemistry. Reads were variably trimmed at the 3′ end based on the Illumina Read Segment Quality Indicator (Illumina manual), and read pairs containing ambiguous bases were removed. Read mapping onto the K10′ genome sequence was performed using SHRiMP (ver. 1.3.2) (2), and single-nucleotide polymorphisms and indels (deletion and insertion polymorphisms [DIPs]) were called using Nesoni (ver. 0.29; Monash University Victorian Bioinformatics Consortium) with default parameters. Read mapping determined that the data set comprised an average sequence coverage of 72.6 across the K10′ genome. This high sequence coverage allowed differences between K10\K10′ and the resequenced version of the genome, designated K10", to be identified with high confidence.Ninety single-nucleotide differences and one 51-bp indel were identified in the K10" genome. As confirmation that these differences are likely to represent errors in the original genome sequence, we have also detected these polymorphisms in two additional bovine M. avium subsp. paratuberculosis genomes recently sequenced and assembled within our laboratory (data not shown). Seven of the 90 differences and the 51-bp indel were subjected to PCR and Sanger sequencing for verification. All of the polymorphisms were confirmed to be present in K10" compared to the original genome sequence.Thirty-six single-nucleotide deletions and four nucleotide insertions were identified in K10" compared to the reference. These DIPs resulted in 27 frameshift mutations of protein coding loci. As a consequence of these frameshifts, one complete coding sequence (CDS) feature was removed (MAPK_3751), one novel CDS was created (MAPK_2081b), and one pseudogene was repaired (MAPK_4158-4159). In almost all of the other cases, the frameshifts resulted in proteins which more closely resembled their orthologs in M. avium subsp. hominissuis and M. intracellulare. Other frameshifts of biological interest include the truncation of a PPE family protein (MAPK_1173) and the extension of an MCE (mammalian cell entry) family protein (MAPK_4086). Compared to the reference, K10" also had a 51-bp indel within a possible MCE family protein (MAPK_1575). This indel consisted of an 11-bp deletion (bases 2436510 to 2436520 in the original K10 sequence) and an insertion of 51 bp. The resulting protein sequence now more closely resembles orthologs of the MCE family in other Mycobacterium spp. In conclusion, the fact that so many of the amended bases have resulted in revised coding regions indicates the underlying importance of this exercise.     

Effects of d-amphetamine on the behavior of pigeons exposed to the peak procedure

The effects of d-amphetamine on pigeons' key-pecking under the peak interval (PI) procedure were investigated in two experiments. In experiment I the effects of doses of d-amphetamine from 0.75 to 3.0 mg/kg on responding under PI 30 and 45 s were studied for 10 successive days. Reductions in peak time and wait time were observed at both PI values and an increase in the width was found at PI 30 s. There was no evidence of tolerance. In experiment II, pigeons exposed to a PI 45 s schedule were administered doses of D-amphetamine of 1.5 and 3.0 mg/kg for 30 successive days. Reductions in peak time and wait time were found here. Evidence of tolerance was found in wait time, peak time and width of the distribution at the higher dose. In both experiments a rate-dependent effect of the drug was found in the portion of each peak trial before the time that food was delivered on reinforced trials; this effect was weaker after the customary time of food delivery. The rate-dependent effect for responses before food time, combined with little effect of the drug on responses after food time, is shown by simulation to be sufficient to account for the reduction in peak time, without the need to appeal to an internal clock mechanism.