Altered fractalkine cleavage results in an organ-specific 17 kDa fractalkine fragment in salivary glands of NOD mice

Sjögren''s syndrome is a rheumatic disease in which the salivary and lacrimal glands are the principal targets of a pathological autoimmune reaction. Previous studies in mice indicated that delayed organogenesis and aberrant cell physiology followed by an increase in acinar cell apoptosis precede chronic focal inflammation in the salivary glands and the manifestation of impaired exocrine gland secretion. In a recent study by Wildenberg and colleagues, the authors report aberrant proteolytic activity in the salivary glands of non-obese diabetic mice and the generation of a unique organ-specific 17 kDa fragment of the chemokine and adhesion molecule fractalkine.In the previous issue of Arthritis Research & Therapy, aberrant proteolytic activity in the salivary glands of non-obese diabetic (NOD) mice with spontaneous experimental Sjögren''s syndrome (SS) was reported [1]. SS is a rather common systemic autoimmune disease characterized by exocrine gland inflammation and impaired glandular function [2]. The NOD strain has become a commonly used spontaneous model for SS in which several SS-related hypotheses have been developed or tested. Although the initiating event leading to the accumulation of mononuclear cells in the exocrine glands is unknown, studies in NOD mice and related congenic strains carrying the Aec1 and Aec2 loci showed aberrant proteolytic activity [3], elevated apoptosis and activated interferon-γ, Toll-like receptor (TLR)3 and TLR7 associated pathways in the salivary glands prior to manifestation of the disease [4].Wildenberg and colleagues [1] now provide evidence that fractalkine is cleaved to a unique organ-specific 17 kDa fragment in the salivary glands of NOD mice. This phenomenon was observed from as early as 10 weeks of age. At this time-point the mice probably displayed a pre-disease or sub-clinical stage of SS [5]. Altered cleavage was subsequently observed until 20 weeks of age when SS in NOD mice is thought to have advanced to an overt disease stage [5]. Unfortunately, the protease involved in the cleavage of this apparently unique and organ-specific17 kDa fragment has not yet been identified. The cleavage, however, did not seem to depend on Caspase-3, ADAM-10, ADAM-17, MMP-2 and/or MMP-9 activity [1]. Throughout the same period of time, NOD mice presented autoantibodies recognizing 31 kDa fractalkine.The authors mainly discuss their finding from the perspective of fractalkine as a potentially new autoantigen in SS [1]. Although such hypotheses are highly speculative considering the present core of knowledge, we believe that chemokines in general, and fractalkine in particular, deserve more attention in SS research. We recently found specific chemokines to be associated with different aspects of experimental SS [6], and prevention of hyposalivation in NOD mice through administration of heat-shock protein 60 kDa coincided with normalization of multiple chemokine levels in saliva [7].In contrast to other chemokines, fractalkine can be found in two specific forms, which allows fractalkine to participate in very distinct biological processes. Soluble fractalkine acts as a potent chemotactic factor for monocytes, natural killer (NK)-cells, and T-cells expressing CX3C receptor (CX3R)1. In addition, a membrane-anchored form, which is unusual for chemokines, is expressed on endothelial cells and also several cell types associated with exocrine glands [8]. To what extent fractalkine expression patterns might be altered in salivary glands obtained from patients with SS in comparison with viral infections or homeostatic conditions, however, remains to be investigated [8].By acting as an adhesion molecule, membrane-bound frac-talkine may facilitate extravasation of CX3CR1-expressing leukocytes [9,10]. In addition, CX3CR1 appears to be a selective surface marker for leukocyte subsets, which exert cytotoxic effector functions. Fractalkine may also lead to increased interferon-γ, tumor necrosis factor-γ and granulocyte monocyte colony stimulating factor production by NK-cells and other cell subsets that have been suggested to play a role in the initiation phase and pathogenesis of inflammatory conditions, such as atherosclerosis [10], glomerulonephritis [9] and rheumatoid arthritis (RA) [9]. Although these disorders are multifactorial in nature, exposure to microbial agents is thought to play a role in their initiation [2,9,10]. Several viral proteins were reported to bind a broad spectrum of mediators of the immune system, including fractalkine [8]. Specific gene polymorphisms have been reported to be risk factors for coronary heart disease [10] and deletion of CX3CR1 in apolipoprotein E deficient mice reduced atherosclerotic lesion formation [10]. Fractalkine has also been associated with the pathogenesis of RA after fractalkine and CX3CR1 expression were reported to be upregulated in the synovium of patients with RA [9]. Supporting the notion of the disease-modulating activity of fractalkine in RA, administration of anti-fractalkine antibodies ameliorated experimental RA [9]. With regard to diseases involving the kidneys, a viral fractalkine antagonist reduced kidney inflammation and proteinurea in the Wistar-Kyoto crescentic glomerulonephritis model [9]. In concordance, anti-CX3CR1 blocked lymphocytic infiltration and the development of subsequent stages of glomerulonephritis in these rats [9]. In MRL^lpr mice a truncated fractalkine analogue with the capability of antagonizing the actions of fractalkine also significantly ameliorated several aspects of lupus nephritis and vasculitis [9].The findings reported by Wildenberg and colleagues add the aspect of organ-specific cleavage of fractalkine to its potential role in a specific autoimmune condition. Unfortunately, the report does not address the effect of altered cleavage on fractalkine''s biological activities, for example, chemotaxis. Based on the results presented it is therefore difficult to speculate if fractalkine, through altered cleavage, might be rendered either more potent or less efficient with regard to certain of its actions. The study by Wildenberg and colleagues provides, however, a rationale for conducting such functional studies in the future. In parallel, it would be interesting to address if the described autoantibodies might have the potential to modulate fractalkine related inflammatory processes.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

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.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.