30K Protein Research Articles

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.

Inhibition of apoptosis in HeLa cell by silkworm storage protein 1, SP1

Silkworm contains several distinct proteins that have anti-apoptotic activities in mammalian and insect cells. Although the characteristics of 30K proteins have been well investigated, those of SPs (Storage Proteins) have not been examined. In this article, an anti-apoptotic effect of SP1 was investigated in HeLa cell apoptosis. SP1, a type of insect hemocyanin protein, is a female-specific protein in silkworm and consists of high portion of methionine. Transient expression of SP1 in HeLa cells slightly increased cell viability after the treatment with staurosporine, which induced apoptosis via intrinsic mitochondrial pathway. To investigate the anti-apoptotic effect of SP1, the stable cell line expressing SP1 were screened and constructed. The anti-apoptotic effect of the cell line was assessed using trypan blue exclusion assay, microscopic analysis of nuclear fragmentation, and flow cytometry. SP1 expressing cell line showed strong antiapoptotic activity against the treatment of staurosporine but not TNFa, meaning that SP1 is a protein inhibitor of apoptosis caused by intrinsic apoptosis pathway. As the SP1 is the novel anti-apoptotic protein, the regulation of apoptosis by SP1 will offer a new strategy that can be utilized in biopharmaceutical industry when the focus is on minimization of cell death and maximization of productivity.

Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression.

Sweet orange (Citrus sinensis), one of the most important fruit crops worldwide, may suffer from disease symptoms induced by virus infections, thus resulting in dramatic economic losses. Here, we show that the infection of sweet orange plants with two isolates of Citrus psorosis virus (CPsV) expressing different symptomatology alters the accumulation of a set of endogenous microRNAs (miRNAs). Within these miRNAs, miR156, miR167 and miR171 were the most down-regulated, with almost a three-fold reduction in infected samples. This down-regulation led to a concomitant up-regulation of some of their targets, such as Squamosa promoter-binding protein-like 9 and 13, as well as Scarecrow-like 6. The processing of miRNA precursors, pre-miR156 and pre-miR171, in sweet orange seems to be affected by the virus. For instance, virus infection increases the level of unprocessed precursors, which is accompanied by a concomitant decrease in mature species accumulation. miR156a primary transcript accumulation remained unaltered, thus strongly suggesting a processing deregulation for this transcript. The co-immunoprecipitation of viral 24K protein with pre-miR156a or pre-miR171a suggests that the alteration in the processing of these precursors might be caused by a direct or indirect interaction with this particular viral protein. This result is also consistent with the nuclear localization of both miRNA precursors and the CPsV 24K protein. This study contributes to the understanding of the manner in which a virus can alter host regulatory mechanisms, particularly miRNA biogenesis and target expression.

Identification and Characterization of 30 K Protein Genes Found in Bombyx mori (Lepidoptera: Bombycidae) Transcriptome.

The 30 K proteins, the major group of hemolymph proteins in the silkworm, Bombyx mori (Lepidoptera: Bombycidae), are structurally related with molecular masses of ∼30 kDa and are involved in various physiological processes, e.g., energy storage, embryonic development, and immune responses. For this report, known 30 K protein gene sequences were used as Blastn queries against sequences in the B. mori transcriptome (SilkTransDB). Twenty-nine cDNAs (Bm30K-1–29) were retrieved, including four being previously unidentified in the Lipoprotein_11 family. The genomic structures of the 29 genes were analyzed and they were mapped to their corresponding chromosomes. Furthermore, phylogenetic analysis revealed that the 29 genes encode three types of 30 K proteins. The members increased in each type is mainly a result of gene duplication with the appearance of each type preceding the differentiation of each species included in the tree. Real-Time Quantitative Polymerase Chain Reaction (Q-PCR) confirmed that the genes could be expressed, and that the three types have different temporal expression patterns. Proteins from the hemolymph was separated by SDS-PAGE, and those with molecular mass of ∼30 kDa were isolated and identified by mass spectrometry sequencing in combination with searches of various databases containing B. mori 30K protein sequences. Of the 34 proteins identified, 13 are members of the 30 K protein family, with one that had not been found in the SilkTransDB, although it had been found in the B. mori genome. Taken together, our results indicate that the 30 K protein family contains many members with various functions. Other methods will be required to find more members of the family.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/β transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

Leucine residues in conserved region of 33K protein of bovine adenovirus – 3 are important for binding to major late promoter and activation of late gene expression

The L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37kDa in BAdV-3 infected cells and one protein of 42kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP.

Abstract 423: CC-Chemokine Class Inhibition Attenuates Inflammatory Induced Pathological Angiogenesis Whilst Preserving Ischaemia Driven Physiological Angiogenesis

Introduction: Angiogenesis is critical for survival and in the regenerative response to tissue hypoxia. An imbalance in its regulation causes excessive angiogenesis, exacerbating inflammatory diseases such as cancer and atherosclerosis. Increasing evidence suggests the CC-chemokine class promote inflammatory-driven angiogenesis but, in contrast, have no role in hypoxia-driven angiogenesis. Inhibition of the CC-chemokine class may therefore regulate angiogenesis differently depending on the pathophysiological context. Aim: Using the broad-spectrum CC-chemokine inhibitor ‘35K’, compare the role of the CC-chemokine class in inflammatory versus ischaemia driven angiogenesis. Results: Adenoviruses expressing GFP (AdGFP) or 35K (Ad35K) were injected into mice 3 days prior surgery. Using the murine femoral cuff model of inflammatory angiogenesis, we found a significant decrease in adventitial capillaries (39.7%) and arterioles (75.5%) in the Ad35K group compared to the AdGFP controls, p<0.05. Ad35K mice also had significantly less macrophages (CD68, 32.5%). In contrast, 35K had no effect on markers of neovascularisation including: blood flow recovery and hindlimb capillary density, in the murine hind limb ischaemia model. Consistent with our in vivo findings, in vitro angiogenic assays with endothelial cells, revealed that 35K protein had inhibitory effects on inflammation-induced tubule formation (95.8%), proliferation (88.3%) and migration (15.8%), p<0.05. In hypoxia the inhibitory effects of 35K were more modest for tubule formation (45.2%) and proliferation (43.8%), and migration was completely preserved. Under inflammatory conditions 35K inhibited VEGF (53.4%) and HIF-1α (87.3%) cell protein levels, yet in hypoxia 35K had no effect on VEGF and had a more modest inhibitory effect on HIF-1α (65.4%). Conclusion: Broad-spectrum CC-chemokine inhibition by 35K inhibits inflammation-induced angiogenesis, whilst preserving ischaemia-driven angiogenesis in vitro and in vivo. CC-Chemokine inhibition may be therapeutic strategy to reduce pathological angiogenic diseases without the severe side effects of current therapies caused by complete inhibition in both contexts.

Abstract 630: Broad-spectrum Inhibition of the CC-Chemokine Class Accelerates Wound Healing.

Introduction: Wound healing is a multistep process involving inflammation, proliferation and remodelling at the wound site. Macrophages are important in the inflammatory stage to remove debris, however excessive macrophage accumulation may prolong the inflammatory response leading to prolonged wound healing, excessive scar formation and loss of function at the injury site. In the wound healing process, the CC-chemokine class is involved in the recruitment of macrophages to the wound site. Inhibition of the CC-chemokine class to reduce macrophage infiltration may present as strategy to increase wound closure and reduce scar formation. Aim: To determine if broad spectrum CC-chemokine inhibitor, 35K, can improve wound healing by reducing the inflammatory response and preventing scar formation. Results: Two full-thickness wounds were created on the dorsum, one each side of the midline, of C57BL6 mice. Wounds were treated topically with PBS or 35K protein daily for 10 days. Wounds treated with 35K closed significantly faster than PBS treated wounds between days 4 - 6 (p<0.05) post-wounding. Laser Doppler measurements revealed 35K significantly increased blood flow at the early (day 3-4) and late stages (day 10) of wound repair. Ten days after wounding, collagen was significantly lower in the 35K treated wounds (25.3% p<0.05). There were also reductions in neovessels (CD31, 39%, p<0.001), arterioles (alpha actin, 48% p<0.01) and macrophages (CD68, 25%), as a percentage of wound area, in 35K treated wounds. In wound tissue collected on day 4 (early stage) post-wounding, 35K treatment had an inhibitory effect on CCL2 and CCL5 protein by 41% (p<0.05) and 36% (p<0.05) respectively, and were reduced by 22% and 66% (p<0.05) respectively at day 10 (late stage) wound healing. Consistent with this, mRNA levels of p65 also decreased in both day 4 (34%, p<0.05) and day 10 (62%, p<0.05) wounds treated with 35K. Conclusion: The CC-chemokine class appears to play a key role in wound repair as topical application of CC-chemokine inhibitor 35K promoted wound healing. This was likely to be via inhibition of inflammation and promotion of blood flow recovery in the early stages of wound repair, thereby resulting in less scar formation.

Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts.

Abstract A38: Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R

Abstract Over 50% percent of melanomas carry activating BRAF mutation with a vast majority of these mutations (>90%) resulting from a single nucleotide switch at amino acid 600 resulting in a glutamic acid substitution for valine (V600E). As a result of this mutation BRAF Kinase is constitutively expressed and results in disregulation of the RAS-RAF-MEK-ERK protein kinase pathway that controls cell proliferation and apoptosis. Although, Vemurafenib, a specific inhibitor of the V600E BRAF mutant, has shown to have initial positive clinical response in clinical trials, drug resistant develops after several months of continuous exposure resulting in the recurrence of the treated cancer. We have developed a modified adenovirus vector (AdILV-19) that effectively kills vemurafenib resistant cells. We demonstrate that AdILV-19 suppresses expression of c-Myc, survivin, cyclin D1, B-catenin, and Mcl-1 in BRAF V600E mutated cells that are resistant to vemurafenib. This novel vector, AdILV-19 is derived from our previously published adenoviral vector TAV-255 through an additional modification of the adenoviral genome. In order to facilitate and maximize recombinant adenoviral induced apoptosis by this new vector, we removed the first 200 base pair of the adenoviral E1b 19k protein, a human BCL-2 homolog. Using this newly modified virus in two highly Vemurafenib resistant melanoma cell lines (451lu-R, Mel1617-R) we were able to not only infect and express viral proteins i.e. E1a, but also induce a significant cell killing in these resistant BRAF mutant cell lines by MTT as well as crystal violet assays. MTT assay showed 35% to 45% cell killing at an MOI as low as 15 to 80 to 90% killing at an MOI of 50. Crystal violet assay on the other hand showed a significant cytotoxicity in these resistant cells at a low MOI of 15. Additionally, we were able to show using Immunoblot analysis a postinfection reduction in the expression of major pro-survival proteins in time dependent manner. We observed as early as 24hrs postinfection c-Myc (an oncogenic transcription factor) and survivin (Pro-survival protein) expression were turned off while expression of cyclin D1 (a regulatory subunit of holoenzyme) which shown to be amplified in BRAF inhibitor resistant melanoma cells and Mcl-1 (a pro-survival protein) were turned off as early as 48hs postinfection in both 451lu-R and Mel1617-R melanoma cells. Furthermore, colony formation assays showed a substantial reduction in the ability of single cells to form colonies after infection at MOI of 20 with AdILV-19 in both of these resistant melanoma cell lines. As an extension of these studies we also studied the activity of AdILV-19 to treat a xenograft nude mouse model with subcutaneous injection of 451lu-R melanoma cells. When the tumor volume reached 100 mm3 the mice received 3 intratumoral injections at an MOI of 20. Following treatment, we observed a significant inhibition of tumor growth (p≤0.02) in treated mice (n=4) when compared with PBS treated mice controls. Taken together these data we show this newly modified adenovirus vector AdILV-19 alters and silences cellular oncogenic protein synthesis in vemurafenib resistant BRAF mutant melanomas and has a great potential as therapeutic for the treatment of these human resistant melanomas. Citation Format: Farah Hedjran, Lingzhi Zhang, Jason Sicklick, Tony Reid. Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A38.

Contrasting in vitro vs. in vivo effects of a cell membrane-specific CC-chemokine binding protein on macrophage chemotaxis.

Chemokines (CK) provide directional cues that mediate the recruitment of leukocytes to sites of inflammation. Broad-spectrum blockade of the CC-CK family, using the vaccinia virus 35K protein, has been shown to cause a potent reduction of systemic inflammation in models of atherosclerosis, vein graft disease and arthritis. We have used a cell membrane-targeted form of 35K, Mem35K, to probe whether cell-associated blockade of chemokine response is sufficient to reduce cell recruitment in inflammation. In Tie2cre mice, activation of a flox-stop Mem35K transgene directed conditional expression of Mem35K in leukocytes and endothelial cells, confirmed by Western blotting, flow cytometry and immunofluorescence microscopy. This conditional Mem35K expression was sufficient to increase cell surface CCL5 binding and reduce chemotaxis in vitro to CCL5, CCL2 and CCL3 but not to non-CC-CK chemoattractants, LTB4, C5a or chemerin. However, in vivo monocyte recruitment into the peritoneum driven by zymosan or CC-chemokine injection, which was demonstrated to be CC-CK dependent using CCR2−/− mice, was not reduced by Mem35K expression, despite the expression of functional Mem35K protein. These findings highlight differing requirements for cell-associated anti-inflammatory activity in in vitro and in vivo models.Key message Mem35K is a cell-associated CC-chemokine binding protein.Conditional Mem35K transgenic mice show expression Mem35K in leukocytes.Mem35K blocks in vitro primary macrophage chemotaxis specifically towards CC-chemokines.Mem35K expression is not sufficient to reduce inflammation in vivo.The requirements for anti-inflammatory activity in vitro and in vivo are different. Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-014-1194-6) contains supplementary material, which is available to authorized users.

Conserved arginines of bovine adenovirus-3 33K protein are important for transportin-3 mediated transport and virus replication.

The L6 region of bovine adenovirus (BAdV)-3 encodes a spliced protein designated 33K. The 33K specific sera detected five major proteins and three minor proteins in transfected or virus infected cells, which could arise by internal initiation of translation and alternative splicing. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells. The 33K nuclear transport utilizes both classical importin-α/-β and importin-β dependent nuclear import pathways and preferentially binds to importin-α5 and transportin-3 receptors, respectively. Analysis of mutant 33K proteins demonstrated that amino acids 201–240 of the conserved C-terminus of 33K containing RS repeat are required for nuclear localization and, binding to both importin-α5 and transportin-3 receptors. Interestingly, the arginine residues of conserved RS repeat are required for binding to transportin-3 receptor but not to importin-α5 receptor. Moreover, mutation of arginines residues of RS repeat proved lethal for production of progeny virus. Our results suggest that arginines of RS repeat are required for efficient nuclear transport of 33K mediated by transportin-3, which appears to be essential for replication and production of infectious virion.

A 6K-deletion variant of salmonid alphavirus is non-viable but can be rescued through RNA recombination.

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.

Organ-specific proteomic analysis of NaCl-stressed germinating soybeans.

A comparative proteomic approach was employed to explore proteome expression patterns in germinating soybeans under NaCl stress and NaCl-aminoguanidine treatment. The proteins were extracted from 4-day-old germinating soybean cotyledons and noncotyledons (hypocotyl and radicle) and were separated using two-dimensional polyacrylamide gel electrophoresis. A total of 63 and 72 differentially expressed proteins were confidently identified by MALDI-TOF/TOF in the noncotyledons and cotyledons, respectively. These identified proteins were divided into ten functional groups and most of them were predicted to be cytoplasmic proteins in noncotyledons. Moreover, γ-aminobutyric acid was accumulated while the major allergen (Bd 30K protein) was reduced in the germinating soybeans. The proteins involved in energy metabolism and in protein processing in endoplasmic reticulum were enriched under NaCl stress. Meanwhile, the negative effect of stress was aggravated once polyamine degradation was inhibited. Redistribution of storage proteins under stress indicated that storage proteins might not only function as seed storage reserves but also have additional roles in plant defense.

Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3.

30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3.

Formation and intracellular movement of cytoplasmic bodies of the tomato mosaic virus 126-kDa replication protein in association with the viral movement protein

During plant infection by tobamoviruses such as Tomato mosaic virus (ToMV), viral replication complexes containing a viral 126-kDa replicase component (126K) are generated, temporarily in association with a viral movement protein (MP) that is indispensable for viral intercellular movement. The tobamovirus 126K protein has been shown to play an important role not only in replication but also in intercellular transport of viral RNAs. Transient expression of the 126K protein has recently been reported to result in formation of cytoplasmic bodies with shapes similar to those of the replication complexes. In this study, to investigate the roles of ToMV MP in the formation, localization, and dynamics of cytoplasmic bodies of the 126K protein, we carried out confocal microscopy studies of fluorescently tagged 126K and MP that were transiently and simultaneously expressed in Nicotiana benthamiana. Without MP expression, 126K was evenly distributed in the cytoplasm, with occasional formation of small 126K bodies. These small 126K bodies moved in the cytoplasmic stream and along the endoplasmic reticulum network. In contrast, co-expression of MP markedly promoted the formation of larger MP-associated 126K bodies, which were generally static but moved occasionally in the cytoplasmic stream. In addition, we found that small, mobile 126K bodies were captured at the terminal part of MP-derived filamentous structures, leading to the formation of 126K-MP complexes and their translocation to another cellular site.

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography-tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49%) were predicted to function as binding proteins and 46% were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.

Adenovirus E1B 19-kilodalton protein modulates innate immunity through apoptotic mimicry.

Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not repress macrophage proinflammatory responses and enhanced some cytokine responses. Our results define a new function of the antiapoptotic, adenoviral protein E1B 19K, which we have termed "apoptotic mimicry." Our studies suggest the possibility that the presence or absence of this E1B 19K function could alter the immunological outcome of both natural and therapeutic adenoviral infections. For example, emerging, highly immunopathogenic adenovirus serotypes might induce increased host inflammatory responses as a result of altered E1B 19K function or expression. It is also possible that engineered variations in E1B 19K expression/function could be created during adenovirus vector design that would increase the therapeutic efficacy of replicating adenovirus vectors for vaccines or oncolytic viral targeting of neoplastic cells.

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance for growth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, and regulating hormones, and they have been identified using proteomic approaches. However, detailed differential expression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterized the differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry identification and bioinformatics methods. We extracted the fat body proteins from 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning [LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11 important sex-specific expression proteins and 14 stage-specific expression proteins. We accurately identified 13 of these specific expression proteins, including actin, calponin-like protein, 75kDa subunit NADH, receptor for activated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin 1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30K protein precursor. We showed that BmRACK was differentially expressed between male and female silkworms. We discuss the biological roles of the specific expression proteins during the larval–pupal developmental stages.

Differential Expression of Stage-Specific Fat Body Proteins during Larval-Pupal Period in Silkworm (<i>Bombyx mori</i>)

The silkworm fat body is the organ of many intermediary metabolic processes and sustenance of growth throughout the life, and its proteins are responsible for storing nutrients, providing energy and regulating hormones. To date, detailed differential expressions of fat body proteins have not been clearly analyzed. In this research, fat body proteins were extracted from the larvae of the 5th day of the 5th instar, un-pupated larvae of spinning end and pupae of pupating day of the multivoltine silkworm variety Da Zao, and the differential expressed proteins were characterized by two-dimensional gel electrophoresis, followed by mass spectrometry identification and informatics method. 14 stage-specific expression proteins were detected and confirmed. Among them, 7 proteins were accurately identified, such as actin, calponin-like protein, beta-tubulin, hypothetical protein, antichymotrypsin precursor and 30K protein precursor etc. The biological roles of these important proteins during the larval-pupal developmental stages were discussed.