The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

Simplified construction and performance of a device for particle bombardment

A modified particle inflow gun (PIG), that utilized a plastic vacuum chamber, was compared with a conventional PIG by bombarding cantaloupe (Cucumis melo L.) cotyledon basal quarters with plasmid pBI221 (Clontech Inc.) containing a -glucuronidase (GUS) gene adsorbed onto tungsten particles. Both guns produced an equivalent number of transient GUS foci when tested at 410 kPa (60 psi), 620 kPa (90 psi) or 830 kPa (120 psi) helium and at a 10, 15 or 20 cm gap between the specimen and DNA/particle holder screen. For both guns, treatments utilizing the lower pressure and/or the greater distance generally produced significantly fewer GUS-positive foci. The plastic PIG was convenient to operate and could be built with simple hand tools in less than 40 minutes, using readily available parts.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Inactivation of monoamine oxidase A by the monoamine oxidase B inactivators 1-phenylcyclopropylamine, 1-benzylcyclopropylamine, and N-cyclopropyl-alpha-methylbenzylamine

Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Background

The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.

Results

The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.

Conclusions

Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.