Mary Ann Gawinowicz

Identification of the alpha chain lysine donor sites involved in factor XIIIa fibrin cross-linking.

Biochemical studies of fibrin cross-linking were conducted to identify the specific Aα chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during α polymer formation. A previously characterized Factor XIIIa fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH2-terminal cross-linking domain of α2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five α chain CNBr fragments (within Aα 208-610) and one γ chain CNBr fragment (γ 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an α chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within α 208-610 had incorporated the peptide probe, whereas γ chain donor activity was due solely to peptide cross-linking at (γ) Lys406; the α chain lysines, Lys556 and Lys580, accounted for 50% of the total α chain donor cross-linking activity observed, with Lys539, Lys508, Lys418, and Lys448 contributing an additional 28% and Lys601, Lys606, Lys427, Lys429, Lys208, Lys224, and/or Lys219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of α polymer formation, raise questions concerning the physiological role of multiple α chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin α chain cross-linking.

A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex.

To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.

Identification of Potential Tumor Differentiation Factor (TDF) Receptor from Steroid-responsive and Steroid-resistant Breast Cancer Cells

Background: Tumor differentiation factor (TDF) is a newly identified pituitary protein with no known receptor. Results: Heat shock 70-kDa proteins are potential TDF receptor candidates. Conclusion: TDF acts on breast cells through a novel pathway. Significance: These data may help to elucidate the role of TDF. Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Mass Spectrometric Evidence That Proteolytic Processing of Rainbow Trout Egg Vitelline Envelope Proteins Takes Place on the Egg

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEα,VEβ, and VEγ, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEα-VEγ and VEβ-VEγ were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.

Disulfide proteomics for identification of extracellular or secreted proteins.

The combination of SDS‐PAGE and MS is one of the most powerful and perhaps most frequently used gel‐based proteomics approaches in protein identification. However, one drawback of this method is that separation takes place under denaturing and reducing (R) conditions and as a consequence, all proteins with identical apparent molecular mass (Mr) will run together. Therefore, low‐abundant proteins may not be easily identified. Another way of investigating proteins by proteomics is by analyzing subproteomes from a total proteome such as phosphoproteomics, glycoproteomics, or disulfide proteomics. Here, we took advantage of the property of secreted proteins to form disulfide bridges and investigated disulfide‐linked proteins, using SDS‐PAGE under nonreducing (NR) conditions. We separated sera from normal subjects and from patients with various diseases by SDS‐PAGE (NR) and (R) conditions, followed by LC‐MS/MS analysis. Although we did not see any detectable difference between the sera separated by SDS‐PAGE(R), we could easily identify the disulfide‐linked proteins separated by SDS‐PAGE (NR). LC‐MS/MS analysis of the disulfide‐linked proteins correctly identified haptoglobin (Hp), a disulfide‐linked protein usually found as a heterotetramer or as a disulfide‐linked heteropolymer. Western blotting under NR and R conditions using anti‐Hp antibodies confirmed the LC‐MS/MS experiments and further confirmed that upon reduction, the disulfide‐linked Hp heterotetramers and polymers were no longer disulfide‐linked polymers. These data suggest that simply by separating samples on SDS‐PAGEunder NR conditions, a different, new proteomics subset can be revealed and then identified.

Identification of a potential tumor differentiation factor receptor candidate in prostate cancer cells

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the bloodstream and has an endocrine function. TDF and TDF‐P1, a 20‐residue peptide selected from the ORF of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF has no known mechanism of action. In our recent study, we identified heat shock 70 kDa proteins (HSP70s) as TDF receptors (TDF‐Rs) in breast cancer cells. Therefore, we sought to investigate whether TDF‐R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF‐P1 to purify the potential TDF‐R candidates by affinity purification chromatography from DU145 and PC3 steroid‐resistant prostate cancer cells, LNCaP steroid‐responsive prostate cancer cells, and nonprostate NG108 neuroblastoma and BLK CL.4 fibroblast‐like cells. We identified the purified proteins by MS, and validated them by western blotting, immunofluorescence microscopy, immunoaffinity purification chromatography, and structural biology. We identified seven candidate proteins, of which three were from the HSP70 family. These three proteins were validated as potential TDF‐R candidates in LNCaP steroid‐responsive and in DU145 and PC3 steroid‐resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast‐like cells. Our previous study and the current study suggest that GRP78, and perhaps HSP70s, are strong TDF‐R candidates, and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells, inducing cell differentiation through a novel, steroid‐independent pathway.

Photoaffinity labeling of rhodopsin and bacteriorhodopsin.

Photoaffinity labeling with bovine rhodopsin using a retinal with a fixed 11-cis-ene cross-linked exclusively to Trp-265/Leu-266 in helix F, showing that the beta-ionone C-3 is close to helix F. Moreover, since these labeled amino acids are in the middle of helix F, while the Schiff-base linkage to Lys-296 at the other terminus of the chromophore is also in the middle of helix G, the chromophore lies horizontally near the center of the lipid bilayer. In bacteriorhodopsin, photoaffinity studies using a retinal with a C-10 tritiated phenylazide appended through a 13 A spacer cross-linked to Arg-175/Asn-176 on the cytoplasmic side of helix F; this indicates that 9-Me points toward the extracellular space. This result agrees with our earlier studies with 9-sulfate analogs but is opposite to that deduced by biophysical measurements.

Characterization of tumor differentiation factor (TDF) and its receptor (TDF-R)

Tumor differentiation factor (TDF) is an under-investigated protein produced by the pituitary with no definitive function. TDF is secreted into the bloodstream and targets the breast and prostate, suggesting that it has an endocrine function. Initially, TDF was indirectly discovered based on the differentiation effect of alkaline pituitary extracts of the mammosomatotropic tumor MtTWlO on MTW9/PI rat mammary tumor cells. Years later, the cDNA clone responsible for this differentiation activity was isolated from a human pituitary cDNA library using expression cloning. The cDNA encoded a 108-amino-acid polypeptide that had differentiation activity on MCF7 breast cancer cells and on DU145 prostate cancer cells in vitro and in vivo. Recently, our group focused on identification of the TDF receptor (TDF-R). As potential TDF-R candidates, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. Here we review the current advances on TDF, with particular focus on the structural investigation of its receptor and on its functional effects on breast and prostate cells.

DOUBLE POINT CHARGE MODEL FOR VISUAL PIGMENTS; EVIDENCE FROM DIHYDRORHODOPSINS*

The visual pigment rhodopsin is known to consist of a chromophore, 1 1 4 s retinal 1 bound to the terminal amino group of a lysine moiety of the apoprotein opsin. It is also generally accepted that the linkage to the protein is through a protonated Schiff base (Shriver et al., 1977).

Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

The action of light on the pigment causes the 1 1 4 s chromophore to isomerize and become detached from the protein, releasing free allfruns retinal and opsin at the end of the bleaching sequence. A fundamental problem arises when the absorption maximum of the protonated Schiff base formed from simple amines such as butylamine (2) absorb at 440nm in the leveling solvent methanol (Blatz, 1968),‘, whereas when R is protein (3), the maxima range from 430 to 580nm depending on the source of opsin. Typically for bovine rhodopsin the