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Biomedical subjects

Scott C Mohr

Publications and source records attributed to Scott C Mohr.

5 recordsLinked to original sources
PubMed2005

Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein.

Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.

Affinity Labels↗
PubMed2004

Ribosomal protein-sequence block structure suggests complex prokaryotic evolution with implications for the origin of eukaryotes.

Amino acid sequence alignments of orthologous ribosomal proteins found in Bacteria, Archaea, and Eukaryota display, relative to one another, an unusual segment or block structure, with major evolutionary implications. Within each of the prokaryotic phylodomains the sequences exhibit substantial similarity, but cross-domain alignments break up into (a) universal blocks (conserved in both phylodomains), (b) bacterial blocks (unalignable with any archaeal counterparts), and (c) archaeal blocks (unalignable with any bacterial counterparts). Sequences of those eukaryotic cytoplasmic riboproteins that have orthologs in both Bacteria and Archaea, exclusively match the archaeal block structure. The distinct blocks do not correlate consistently with any identifiable functional or structural feature including RNA and protein contacts. This phylodomain-specific block pattern also exists in a number of other proteins associated with protein synthesis, but not among enzymes of intermediary metabolism. While the universal blocks imply that modern Bacteria and Archaea (as defined by their translational machinery) clearly have had a common ancestor, the phylodomain-specific blocks imply that these two groups derive from single, phylodomain-specific types that came into existence at some point long after that common ancestor. The simplest explanation for this pattern would be a major evolutionary bottleneck, or other scenario that drastically limited the progenitors of modern prokaryotic diversity at a time considerably after the evolution of a fully functional translation apparatus. The vast range of habitats and metabolisms that prokaryotes occupy today would thus reflect divergent evolution after such a restricting event. Interestingly, phylogenetic analysis places the origin of eukaryotes at about the same time and shows a closer relationship of the eukaryotic ribosome-associated proteins to crenarchaeal rather than euryarchaeal counterparts.

Amino Acid Sequence↗
PubMed2003

CK2 phosphorylation of the armadillo repeat region of beta-catenin potentiates Wnt signaling.

Protein kinase CK2 is a ubiquitous serine/threonine kinase involved in many biological processes. It is overexpressed in many malignancies including rodent and human breast cancer, and is up-regulated in Wnt-transfected mammary epithelial cells, where it can be found in a complex with dishevelled and beta-catenin. beta-Catenin is a substrate for CK2 and inhibition of CK2 reduces levels of beta-catenin and dishevelled. Here we report that inhibition of CK2 using pharmacologic agents or expression of kinase inactive subunits reduces beta-catenin-dependent transcription and protein levels in a proteasome-dependent fashion. The major region of phosphorylation of beta-catenin by CK2 is the central armadillo repeat domain, where carrier proteins like axin and the adenomatous polyposis coli gene product APC interact with beta-catenin. The major CK2 phosphorylation site in this domain is Thr393, a solvent-accessible residue in a key hinge region of the molecule. Mutation of this single amino acid reduces beta-catenin phosphorylation, cotranscriptional activity, and stability. Thus, CK2 is a positive regulator of Wnt signaling through phosphorylation of beta-catenin at Thr393, leading to proteasome resistance and increased protein and co-transcriptional activity.

Animals↗
PubMed2002

An estradiol-porphyrin conjugate selectively localizes into estrogen receptor-positive breast cancer cells.

A conjugate of a C(11)-beta-derivative of estradiol and an asymmetric tetraphenylporphyrin was synthesized to study its potential selective uptake by breast cancer cells naturally over-expressing the nuclear receptor for estrogen (ER). Competitive radioligand binding assays of this conjugate with recombinant ER showed that the conjugate bound to ER in a dose-dependent manner with an EC50 of 274 nM, compared with 1 nM for estradiol, the natural ligand. Cellular uptake studies with ER-positive MCF-7 and ER-negative HS578t human breast cancer cells revealed that, the conjugate was taken up by MCF-7 cells in a dose-dependent manner, which was obliterated by co-incubation with a large excess of estradiol. On the other hand there was very little uptake of the un-conjugated porphyrin by MCF-7 and Hs578t cells. HS578t cells also showed insignificant uptake of the conjugate under the conditions of our experiment. These results strongly suggested that specific interaction between the endogenous ER in MCF-7 cells and the estrogen part of the conjugate enabled these cells to selectively internalize the conjugate over the un-conjugated porphyrin. Therefore, ER-binding conjugates of estradiol and porphyrins could potentially be used for ER-targeted photodynamic therapy of hormone-sensitive cancers of breast, ovary, gonads etc.

Antineoplastic Agents↗