Dr. David Rimm is interviewed by Feras Akbik

Dr. David Rimm, MD PhD, is a professor of Pathology at the Yale University School of Medicine specializing in developing quantitative, diagnostic techniques. His lab recently engineered a fluorescence-based algorithm, Automated Quantitative Analysis (AQUA), to analyze tissue microarrays in the hope of moving toward personalized medicine and diagnoses.     

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.     

Lack of association between receptor protein tyrosine phosphatase RPTP mu and cadherins

RPTP mu is a receptor-like protein tyrosine phosphatase that mediates homophilic cell-cell interactions. Surface expression of RPTP mu is restricted to cell-cell contacts and is upregulated with increasing cell density, suggesting a role for RPTP mu in contact-mediated signaling. It was recently reported (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977-986) that RPTP mu binds directly to cadherin/catenin complexes, and thus may regulate the tyrosine phosphorylation of such complexes. Here we report that this concept needs revision. Through reciprocal precipitations using a variety of antibodies against RPTP mu, cadherins, and catenins, we show that RPTP mu does not interact with cadherin/catenin complexes, even when assayed under very mild lysis conditions. We find that the anti- RPTP mu antiserum used by others precipitates cadherins in a nonspecific manner independent of RPTP mu. We conclude that, contrary to previous claims, RPTP mu does not interact with cadherin complexes and thus is unlikely to directly regulate cadherin/catenin function.     

Dynamic Interaction of PTPμ with Multiple Cadherins In Vivo

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites

We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7- nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.     

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.     

Proteomic characterization of herbicide safener-induced proteins in the coleoptile of Triticum tauschii seedlings

Proteomic methods such as two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry, as well as immunoblotting, were used to identify herbicide safener-induced proteins in the coleoptile of Triticum tauschii, a diploid wheat containing the D genome also found in the cultivated, hexaploid wheat Triticum aestivum. The herbicide safener fluxofenim dramatically increased protein abundance in the molecular weight (M(r)) range of 24 to 30 kDa, as well as a few higher M(r) proteins, in the coleoptile of T. tauschii seedlings. In total, twenty proteins were identified in this study. Eleven proteins were highly safener induced and only weakly expressed in the control; seven proteins were new safener induced proteins that were not detected in the control. Two other proteins were constitutively expressed in both the control and safener-treated coleoptiles. Among the eighteen inducible proteins, fifteen were glutathione S-transferase (GST) subunits that fall into three subclasses: eight proteins were from the tau subclass, six proteins were from the phi subclass, and one protein was from the lambda class. Another three safener inducible proteins showed homology to the aldo/keto reductase family and with proteins that have roles in glycolysis and the Krebs cycle. Two constitutively expressed proteins were identified, one having highest homology to the dehydroascorbate reductase subclass of GSTs and one with an ascorbate peroxidase. Immunoblot analyses, using two different antisera raised against the same GST protein but differing in their specificity, were used to further characterize the GST proteins expressed in response to safener treatment. Results from immunoblotting, combined with mass spectral analysis, showed that post-translational modification of GST proteins in control and safener-treated coleoptiles may occur.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Isolation and characterization of bacterial flagellar hook proteins from salmonellae and Escherichia coli.

Flagellar hook proteins from Salmonella and Escherichia coli were dissociated in acid and purified by diethylamino-ethyl-cellulose column chromatography. These two proteins had the same electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. However, analytical electrofocusing patterns showed that these proteins had different isoelectric points (4.7 for Salmonella typhimurium and 4.4 for E. coli). Immunodiffusion and immuno-electron microscopy carried out with antisera prepared against purified hook proteins from S. typhimurium and E. coli showed that these antisera reacted with both hooks. Affinity chromatography allowed separation of antibodies specific for hook proteins from each bacterial species. These results indicate that the hook proteins share common antigenic determinants as well as specific antigens, although the specificity is not quantitatively resolved. From comparisons of the amino acid composition of the hook proteins and flagellins, it was concluded that the differences between flagellins from S. typhimurium and E. coli were larger than those between hook proteins from these species.     

Analysis of cytoplasmic membrane proteome of Streptococcus pneumoniae by shotgun proteomic approach

In this study, cytoplasmic membrane proteins of S. pneumoniae strain R6 (ATCC BBA-255) were effectively separated from cell wall or extracellular proteins by sodium carbonate precipitation (SCP) and ultracentrifugation. Forty seven proteins were analyzed as cytoplasmic membrane proteins from the 260 proteins identified by the shotgun proteomic method using SDS-PAGE/LC/MS-MS. ABC transporters for metabolites such as metals, oligopeptides, phosphate, sugar, and amino acids, and membrane proteins involved in phosphotransferse systems, were identified as the predominant and abundant, cytoplasmic membrane proteins that would be essential for nutrient uptake, antibiotic resistance and virulence mechanisms. Our result supports that gel-based shotgun proteomics combined with sodium carbonate precipitation and ultracentrifugation is an effective method for analysis of cytoplasmic membrane proteins of S. pneumoniae.     

Proteomic analysis of the secretome of rice calli

The cell wall and extracellular matrix in higher plants include secreted proteins that play critical roles in a wide range of cellular processes, such as structural integrity and biogenesis. Compared with the intensive cell wall proteomic studies in Arabidopsis , the list of cell wall proteins identified in monocot species is lacking. Therefore, we conducted a large-scale proteomic analysis of secreted proteins from rice. Highly purified secreted rice proteins were obtained from the medium of a suspension of callus culture and were analyzed with multidimensional protein identification technology (MudPIT). As a result, we could detect a total of 555 rice proteins by MudPIT analysis. Based on bioinformatic analyses, 27.7% (154 proteins) of the identified proteins are considered to be secreted proteins because they possess a signal peptide for the secretory pathway. Among the 154 identified proteins, 27% were functionally categorized as stress response proteins, followed by metabolic proteins (26%) and factors involved in protein modification (24%). Comparative analysis of cell wall proteins from Arabidopsis and rice revealed that one third of the secreted rice proteins overlapped with those of Arabidopsis . Furthermore, 25 novel rice-specific secreted proteins were found. This work presents the large scale of the rice secretory proteome from culture medium, which contributes to a deeper understanding of the rice secretome.     

The identification and characterization of membranome components

Analyses of proteins from a number of proteomic studies of cell membranes have demonstrated that a significant component of the identified proteins is not predicted to contain transmembrane regions. The presence of such proteins may arise as a result of contamination of the membrane preparations or through real associations. Our aim was to identify integral proteins as well as those that are intimately associated with the microsomal membranes of K562 cells. Isolated membranes were treated under conditions reported to remove noncovalently associated 'peripheral' proteins and the residual proteins were SDS-PAGE-separated and analyzed by LC-MS/MS. Tandem lectin affinity was also examined as a complementary approach for the enrichment of membrane glycoproteins. Approximately 41% of the isolated proteins were assigned as membrane proteins based on the presence of transmembrane regions or covalent post-translational modifications that could account for membrane association. Collectively, these results indicate that there is a significant component of non integral proteins that appear to be as closely associated with membranes as integral elements.     

Use of nonmotile mutants to identify a set of membrane proteins related to gliding motility in Cytophaga johnsonae.

Nonmotile mutants of the gliding bacterium Cytophaga johnsonae were examined to identify proteins that might be involved in gliding motility. Wild-type and mutant cell proteins were solubilized and fractionated by using Triton X-114, and the proteins that partitioned into the aqueous phase or the detergent phase were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for proteins that differed between wild-type and mutant cells. Seventeen proteins, ranging in size from 16 to 150 kilodaltons, were implicated by this technique as having some relationship to gliding and were designated Gld-1 through Gld-17. All Gld proteins behaved as integral membrane proteins, partitioning into the detergent phase. All 56 mutants examined exhibited changes in 1 or more of the Gld proteins, with the number of proteins altered in any mutant varying from 1 to 11. Several lines of evidence suggested that proteins Gld-12 through Gld-15 are glycoproteins. Analysis of banding patterns of detergent-fraction proteins of motile revertants supported the idea that the Gld proteins have a role in gliding motility.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

The membrane proteins of the methanol-induced peroxisome of Candida boidinii. Initial characterization and generation of monoclonal antibodies

Peroxisomes are massively induced when methylotrophic yeasts are cultured on methanol as the sole carbon and energy source. An analysis of the protein composition of the peroxisomal membrane and the generation of probes against two peroxisomal membrane proteins (PMPs) have been undertaken. Peroxisomes from Candida boidinii were obtained from sucrose gradients as previously described or from a novel one-step purification of the organelle on a Percoll gradient. The protein composition of the membranes from these two preparations was virtually identical. About 10 proteins comprise nearly all of its protein mass. The most prominent proteins have molecular masses of 120, 100, 47, 31-32 (a triplet), and 20 kDa; significant amounts of alcohol oxidase and dihydroxyacetone synthase, the two abundant matrix proteins, also remain associated with the membrane. Glycosylation of the membrane proteins could not be detected. Exposure of the membrane to chaotropes shows that PMPs 100 and 20 are the most easily removable, whereas PMP 47 appears to be the most tightly associated. Mice were injected with peroxisomal membrane, and hybridoma lines were isolated that produced antibody against PMP 20, PMP 47, and dihydroxyacetone synthase. Indirect immunofluorescence with these monoclonal antibodies confirmed that all three proteins are localized to the peroxisomal cluster. Immunoblotting experiments demonstrated that peroxisomal membrane as well as matrix proteins are induced by methanol.     

Relative rates of gene fusion and fission in multi-domain proteins

During evolution genes can produce more complex proteins by gene fusion or less complex proteins by gene fission. Considering proteins from 131 completely sequenced genomes from all three kingdoms of life, we identified 2869 groups of multi-domain proteins as a single protein in certain organisms and as two or more smaller proteins with equivalent domain architectures in other organisms. We found that fusion events are approximately four times more common than fission events, and we established that, in most cases, any particular fusion or fission event only occurred once during the course of evolution.     

ERM (Ezrin/Radixin/Moesin)-based Molecular Mechanism of Microvillar Breakdown at an Early Stage of Apoptosis

Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)–induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1β–converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein–based microvillar breakdown in apoptosis will be discussed.     

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.