Engineering Exciton Dynamics with Synthetic DNA Scaffolds.

Excitons are the molecular-scale currency of electronic energy. Control over excitons enables energy to be directed and harnessed for light harvesting, electronics, and sensing. Excitonic circuits achieve such control by arranging electronically active molecules to prescribe desired spatiotemporal dynamics. Photosynthetic solar energy conversion is a canonical example of the power of excitonic circuits, where chromophores are positioned in a protein scaffold to perform efficient light capture, energy transport, and charge separation. Synthetic systems that aim to emulate this functionality include self-assembled aggregates, molecular crystals, and chromophore-modified proteins. While the potential of this approach is clear, these systems lack the structural precision to control excitons or even test the limits of their power. In recent years, DNA origami has emerged as a designer material that exploits biological building blocks to construct nanoscale architectures. The structural precision afforded by DNA origami has enabled the pursuit of naturally inspired organizational principles in a highly precise and scalable manner. In this Account, we describe recent developments in DNA-based platforms that spatially organize chromophores to construct tunable excitonic systems. The high fidelity of DNA base pairing enables the formation of programmable nanoscale architectures, and sequence-specific placement allows for the precise positioning of chromophores within the DNA structure. The integration of a wide range of chromophores across the visible spectrum introduces spectral tunability. These excitonic DNA-chromophore assemblies not only serve as model systems for light harvesting, solar conversion, and sensing but also lay the groundwork for the integration of coupled chromophores into larger-scale nucleic acid architectures.We have used this approach to generate DNA-chromophore assemblies of strongly coupled delocalized excited states through both sequence-specific self-assembly and the covalent attachment of chromophores. These strategies have been leveraged to independently control excitonic coupling and system-bath interaction, which together control energy transfer. We then extended this framework to identify how scaffold configurations can steer the formation of symmetry-breaking charge transfer states, paving the way toward the design of dual light-harvesting and charge separation DNA machinery. In an orthogonal application, we used the programmability of DNA chromophore assemblies to change the optical emission properties of strongly coupled dimers, generating a series of fluorophore-modified constructs with separable emission properties for fluorescence assays. Upcoming advances in the chemical modification of nucleotides, design of large-scale DNA origami, and predictive computational methods will aid in constructing excitonic assemblies for optical and computing applications. Collectively, the development of DNA-chromophore assemblies as a platform for excitonic circuitry offers a pathway to identifying and applying design principles for light harvesting and molecular electronics.

Photogeneration of Spin Quintet Triplet-Triplet Excitations in DNA-Assembled Pentacene Stacks.

Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems.

Deoxyribonucleic Acid Encoded and Size-Defined π-Stacking of Perylene Diimides.

Natural photosystems use protein scaffolds to control intermolecular interactions that enable exciton flow, charge generation, and long-range charge separation. In contrast, there is limited structural control in current organic electronic devices such as OLEDs and solar cells. We report here the DNA-encoded assembly of π-conjugated perylene diimides (PDIs) with deterministic control over the number of electronically coupled molecules. The PDIs are integrated within DNA chains using phosphoramidite coupling chemistry, allowing selection of the DNA sequence to either side, and specification of intermolecular DNA hybridization. In this way, we have developed a "toolbox" for construction of any stacking sequence of these semiconducting molecules. We have discovered that we need to use a full hierarchy of interactions: DNA guides the semiconductors into specified close proximity, hydrophobic-hydrophilic differentiation drives aggregation of the semiconductor moieties, and local geometry and electrostatic interactions define intermolecular positioning. As a result, the PDIs pack to give substantial intermolecular π wave function overlap, leading to an evolution of singlet excited states from localized excitons in the PDI monomer to excimers with wave functions delocalized over all five PDIs in the pentamer. This is accompanied by a change in the dominant triplet forming mechanism from localized spin-orbit charge transfer mediated intersystem crossing for the monomer toward a delocalized excimer process for the pentamer. Our modular DNA-based assembly reveals real opportunities for the rapid development of bespoke semiconductor architectures with molecule-by-molecule precision.

Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome.

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.

Femtosecond Transient Absorption Microscopy of Singlet Exciton Motion in Side-Chain Engineered Perylene-Diimide Thin Films.

We present a statistical analysis of femtosecond transient absorption microscopy applied to four different organic semiconductor thin films based on perylene-diimide (PDI). By achieving a temporal resolution of 12 fs with simultaneous sub-10 nm spatial precision, we directly probe the underlying exciton transport characteristics within 3 ps after photoexcitation free of model assumptions. Our study reveals sub-picosecond coherent exciton transport (12-45 cm2 s-1) followed by a diffusive phase of exciton transport (3-17 cm2 s-1). A comparison between the different films suggests that the exciton transport in the studied materials is intricately linked to their nanoscale morphology, with PDI films that form large crystalline domains exhibiting the largest diffusion coefficients and transport lengths. Our study demonstrates the advantages of directly studying ultrafast transport properties at the nanometer length scale and highlights the need to examine nanoscale morphology when investigating exciton transport in organic as well as inorganic semiconductors.

Perylene-Based Covalent Organic Frameworks for Acid Vapor Sensing.

Traditionally, the properties and functions of covalent organic frameworks (COFs) are defined by their constituting building blocks, while the chemical bonds that connect the individual subunits have not attracted much attention as functional components of the final material. We have developed a new series of dual-pore perylene-based COFs and demonstrated that their imine bonds can be protonated reversibly, causing significant protonation-induced color shifts toward the near-infrared, while the structure and crystallinity of the frameworks are fully retained. Thin films of these COFs are highly sensitive colorimetric acid vapor sensors with a detection limit as low as 35 µg L-1 and a response range of at least 4 orders of magnitude. Since the acidochromism in our COFs is a cooperative phenomenon based on electronically coupled imines, the COFs can be used to determine simultaneously the concentration and protonation strength of nonaqueous acid solutions, in which pH electrodes are not applicable, and to distinguish between different acids. Including the imine bonds as function-determining constituents of the framework provides an additional handle for constructing multifunctional COFs and extending the range of their possible applications.

Proteomic analysis of Plasmodium falciparum histone deacetylase 1 complex proteins.

Plasmodium falciparum histone deacetylases (PfHDACs) are an important class of epigenetic regulators that alter protein lysine acetylation, contributing to regulation of gene expression and normal parasite growth and development. PfHDACs are therefore under investigation as drug targets for malaria. Despite this, our understanding of the biological roles of these enzymes is only just beginning to emerge. In higher eukaryotes, HDACs function as part of multi-protein complexes and act on both histone and non-histone substrates. Here, we present a proteomics analysis of PfHDAC1 immunoprecipitates, identifying 26 putative P. falciparum complex proteins in trophozoite-stage asexual intraerythrocytic parasites. The co-migration of two of these (P. falciparum heat shock proteins 70-1 and 90) with PfHDAC1 was validated using Blue Native PAGE combined with Western blot. These data provide a snapshot of possible PfHDAC1 interactions and a starting point for future studies focused on elucidating the broader function of PfHDACs in Plasmodium parasites.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

Proteoform-Specific Insights into Cellular Proteome Regulation.

Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.

Oncogenic transformation of lung cells results in distinct exosome protein profile similar to the cell of origin.

Lung cancer is responsible for the highest rate of cancer mortality worldwide. Lung cancer patients are often ineligible for tumor biopsies due to comorbidities. As a result, patients may not have the most effective treatment regimens administered. Patients with mutations in the epidermal growth factor receptor (EGFR) have improved survival in response to EGFR tyrosine kinase inhibitors. A noninvasive method of determining EGFR mutations in patients would have promising clinical applications. Exosomes have the potential to be noninvasive novel diagnostic markers in cancer. Using MS analysis, we identify differentially abundant cell and exosome proteins induced by mutations in p53 and EGFR in lung cells. Importantly, mutations in p53 and EGFR alter cell and exosome protein content compared to an isogenic normal lung epithelial cell. For some proteins, mutation had similar effects in the cell of origin and exosomes. Differences between the cells of origin and exosomes were also apparent, which may reflect specific packaging of proteins into exosomes. These findings that mutations alter protein abundance in exosomes suggest that analysis of exosomes may be beneficial in the diagnosis of oncogenic mutations.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Site-specific glycosylation of the Newcastle disease virus haemagglutinin-neuraminidase.

Members of the Avulavirus, Respirovirus and Rubulavirus genera of the Paramyxoviridae family of viruses utilise haemagglutinin-neuraminidase glycoproteins as their attachment proteins. These glycoproteins are oligomeric type II integral membrane proteins, which possess haemagglutination and sialidase activity. Previous studies have shown that the N-linked glycans present on these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. However, site-specific heterogeneity of these glycans has not been defined. This study concerns characterisation of the glycan compositions attached to haemagglutinin-neuraminidase of the Avulavirus Newcastle disease virus, which causes Newcastle disease in a range of avian species. Haemagglutinin-neuraminidase was derived from egg propagated virions of V4-VAR, an isolate of the avirulent strain QLD/66. Reverse-phase liquid chromatography tandem mass spectrometry strategies including collision induced dissociation, higher-energy collision dissociation and electron-transfer dissociation were implemented to characterise glycopeptides from the haemagglutinin-neuraminidase protein. Overall 63, 58, and 37 glycan compositions were identified at asparagine residues 341, 433 and 481, respectively. N-linked sites 433 and 481 were observed to contain high mannose glycans with paucimannose glycans also observed at site 481. Asparagine residues 341, 433 and 481 contained complex or hybrid glycans with many of the compositions containing variations of fucose and sulfate or phosphate. Sialyation of complex or hybrid N-linked glycans was additionally observed at sites 341 and 433. In addition, a previously undocumented O-linked glycopeptide was identified from the stalk domain of the haemagglutinin-neuraminidase protein. These finding will form the basis for future quantitative glycomic studies of the distribution of glycan structures across N-linked glycosylation sites of Newcastle disease virus haemagglutinin-neuraminidase and assessment of the functional significance of the O-linked glycan in the stalk domain of this protein.

Metagenomic and metaproteomic analyses of Accumulibacter phosphatis-enriched floccular and granular biofilm.

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory-scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high-stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter-enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.

A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection.

Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.

Bovine ephemeral fever rhabdovirus α1 protein has viroporin-like properties and binds importin ß1 and importin 7.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

BACKGROUND: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ßig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFß-induced protein ßig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development.

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.