Biomedical subjects
Ka Wan Li
Publications and source records attributed to Ka Wan Li.
The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences.
It is believed that CLAVATA3 (CLV3) encodes a peptide ligand that interacts with the CLV1/CLV2 receptor complex to limit the number of stem cells in the shoot apical meristem of Arabidopsis thaliana; however, the exact composition of the functional CLV3 product remains a mystery. A recent study on CLV3 shows that the CLV3/ESR (CLE) motif, together with the adjacent C-terminal sequence, is sufficient to execute CLV3 function when fused behind an N-terminal sequence of ERECTA. Here we show that most of the sequences flanking the CLE motif of CLV3 can be deleted without affecting CLV3 function. Using a liquid culture assay, we demonstrate that CLV3p, a synthetic peptide corresponding to the CLE motif of CLV3, is able to restrict the size of the shoot apical meristem in clv3 seedlings but not in clv1 seedlings. In accordance with this decrease in meristem size, application of CLV3p to in vitro-grown clv3 seedlings restricts the expression of the stem cell-promoting transcription factor WUSCHEL. Thus, we propose that the CLE motif is the functional region of CLV3 and that this region acts independently of its adjacent sequences.
Intermittent administration of morphine alters protein expression in rat nucleus accumbens.
Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.
A proteomics approach to identify long-term molecular changes in rat medial prefrontal cortex resulting from sucrose self-administration.
The medial prefrontal cortex (mPFC) is involved in the processing and retrieval of reward-related information. Here, we investigated long-lasting changes in protein composition of the mPFC in rats with a history of sucrose self-administration. Protein levels were analyzed using 2-D PAGE and MALDI-TOF sequencing. From approximately 1500 spots, 28 regulated proteins were unambiguously identified and were involved in cytoskeleton organization, energy metabolism, oxidative stress, neurotransmission, and neuronal outgrowth and differentiation. For several proteins, this change was also found as a long-lasting alteration in gene expression. We show that self-administration of sucrose produces long-lasting molecular neuroadaptations in the mPFC that may be involved in reward-related information processing.
Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons.
Recent studies have begun to focus on the signals that regulate axonal protein synthesis and the functional significance of localized protein synthesis. However, identification of proteins that are synthesized in mammalian axons has been mainly based on predictions. Here, we used axons purified from cultures of injury-conditioned adult dorsal root ganglion (DRG) neurons and proteomics methodology to identify axonally synthesized proteins. Reverse transcription (RT)-PCR from axonal preparations was used to confirm that the mRNA for each identified protein extended into the DRG axons. Proteins and the encoding mRNAs for the cytoskeletal proteins beta-actin, peripherin, vimentin, gamma-tropomyosin 3, and cofilin 1 were present in the axonal preparations. In addition to the cytoskeletal elements, several heat shock proteins (HSP27, HSP60, HSP70, grp75, alphaB crystallin), resident endoplasmic reticulum (ER) proteins (calreticulin, grp78/BiP, ERp29), proteins associated with neurodegenerative diseases (ubiquitin C-terminal hydrolase L1, rat ortholog of human DJ-1/Park7, gamma-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), alpha enolase, aldolase C/Zebrin II) were included among the axonally synthesized proteins. Detection of the mRNAs encoding each of the axonally synthesized proteins identified by mass spectrometry in the axonal compartment indicates that the DRG axons have the potential to synthesize a complex population of proteins. Local treatment of the DRG axons with NGF or BDNF increased levels of cytoskeletal mRNAs into the axonal compartment by twofold to fivefold but had no effect on levels of the other axonal mRNAs studied. Neurotrophins selectively increased transport of beta-actin, peripherin, and vimentin mRNAs from the cell body into the axons rather than changing transcription or mRNA survival in the axonal compartment.
Proteomics of the injured rat sciatic nerve reveals protein expression dynamics during regeneration.
Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the approximately 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, alpha-crystallin B, alpha-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.
Phosphorylation of androgen receptor isoforms.
Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed.
Granularin, a novel molluscan opsonin comprising a single vWF type C domain is up-regulated during parasitation.
Snails are intermediate hosts to schistosome parasites, some of which are the main cause of human schistosomiasis (bilharzia), and have been used as models for parasite-host interactions for a long time. Here, we have characterized a novel internal defense peptide of the snail Lymnaea stagnalis, of which the relative abundance in brain tissue increases upon infection with the avian schistosome Trichobilharzia ocellata. This protein, named granularin, is secreted by granular cells, which are numerous in the connective tissue surrounding the brain. The protein is unique because it comprises only a single Von Willebrand factor type C domain that is normally found in large transmembrane and secreted extracellular matrix proteins. The granularin gene is twice up-regulated during parasitation. Purified granularin stimulates phagocytosis of foreign particles by blood hemocytes. Together, our data indicate that granularin represents a novel protein that acts as an opsonin in the molluscan internal defense response.
Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology.
The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.
Retrograde labeling of single neurons in conjunction with MALDI high-energy collision-induced dissociation MS/MS analysis for peptide profiling and structural characterization.
To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.