Characterization of a Dominant Negative Mutant of the Cell Cycle Ubiquitin-conjugating Enzyme Cdc34

The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme that is required for the cell cycle G(1)/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys and Leu with Ser residues. Cys is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu is highly conserved among all known ubiquitin-conjugating enzymes. Mutants that encode either an alanine or a serine at one or both of these two positions are inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic multibudded phenotype of cdc34 temperature-sensitive or null mutants, this blockade is relieved by simultaneous overexpression of wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to inhibit in vitro ubiquitination of the Cdc34-specific substrate, Cln2 protein. We suggest that Cdc34-C95S,L99S selectively sequesters a subset of Cdc34 substrates or regulators. These findings have implications for the structure/function relationships of ubiquitin-conjugating enzymes, and suggest a general method for identifying components and substrates of specific ubiquitination pathways of eukaryotes

The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands.

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation ofligand from an engaged TCR and (b) recruitment oflck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide-MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-zeta, reduced tyrosine phosphorylation of CD3epsilon, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance

Fourier-Domain Electromagnetic Wave Theory for Layered Metamaterials of Finite Extent

The Floquet-Bloch theorem allows waves in infinite, lossless periodic media to be expressed as a sum of discrete Floquet-Bloch modes, but its validity is challenged under the realistic constraints of loss and finite extent. In this work, we mathematically reveal the existence of Floquet-Bloch modes in the electromagnetic fields sustained by lossy, finite periodic layered media using Maxwell's equations alone without invoking the Floquet-Bloch theorem. Starting with a transfer-matrix representation of the electromagnetic field in a generic layered medium, we apply Fourier transformation and a series of mathematical manipulations to isolate a term explicitly dependent on Floquet-Bloch modes. Fourier-domain representation of the electromagnetic field can be reduced into a product of the Floquet-Bloch term and two other matrix factors: one governed by reflections from the medium boundaries and another dependent on layer composition. Electromagnetic fields in any finite, lossy, layered structure can now be interpreted in the Fourier-domain by separable factors dependent on distinct physical features of the structure. The developed theory enables new methods for analyzing and communicating the electromagnetic properties of layered metamaterials.Comment: 10 pages, 3 figure

The short-lived MATα2 transcriptional regulator is ubiquitinated in vivo

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT-alpha-2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha-2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha-2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha-2 degradation is dependent on its ubiquitination

Relation Between Quantum Speed Limits And Metrics On U(n)

Recently, Chau [Quant. Inform. & Comp. 11, 721 (2011)] found a family of metrics and pseudo-metrics on $n$-dimensional unitary operators that can be interpreted as the minimum resources (given by certain tight quantum speed limit bounds) needed to transform one unitary operator to another. This result is closely related to the weighted $\ell^1$-norm on ${\mathbb R}^n$. Here we generalize this finding by showing that every weighted $\ell^p$-norm on ${\mathbb R}^n$ with 1\le p \le \limitingp induces a metric and a pseudo-metric on $n$-dimensional unitary operators with quantum information-theoretic meanings related to certain tight quantum speed limit bounds. Besides, we investigate how far the correspondence between the existence of metrics and pseudo-metrics of this type and the quantum speed limits can go.Comment: minor amendments, 6 pages, to appear in J.Phys.

The Case for Combining a Large Low-Band Very High Frequency Transmitter With Multiple Receiving Arrays for Geospace Research: A Geospace Radar

We argue that combining a high‐power, large‐aperture radar transmitter with several large‐aperture receiving arrays to make a geospace radar—a radar capable of probing near‐Earth space from the upper troposphere through to the solar corona—would transform geospace research. We review the emergence of incoherent scatter radar in the 1960s as an agent that unified early, pioneering research in geospace in a common theoretical, experimental, and instrumental framework, and we suggest that a geospace radar would have a similar effect on future developments in space weather research. We then discuss recent developments in radio‐array technology that could be exploited in the development of a geospace radar with new or substantially improved capabilities compared to the radars in use presently. A number of applications for a geospace radar with the new and improved capabilities are reviewed including studies of meteor echoes, mesospheric and stratospheric turbulence, ionospheric flows, plasmaspheric and ionospheric irregularities, and reflection from the solar corona and coronal mass ejections. We conclude with a summary of technical requirements