George Korza

Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse.

Role of a SpoVA Protein in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

The proteins encoded by the spoVA operon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA(2,6)) and Ca(2+) into developing spores of the bacterium Bacillus subtilis. The crystal structure of B. subtilis SpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA(2,6) exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA(2,6) and Ca(2+)-DPA(2,6) with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA(2,6)-binding pocket in SpoVAD essentially abolish its DPA(2,6)-binding capacity. Moreover, replacement of the wild-type spoVAD gene in B. subtilis with any of these spoVAD gene variants effectively eliminated DPA(2,6) uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA(2,6) movement into developing B. subtilis spores.

Differential subcellular distributions and trafficking functions of hnRNP A2/B1 spliceoforms.

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11β-hydroxysteroid dehydrogenase, isozyme 1 (11β-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11β-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochromeb5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochromeb5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.

Physical Interaction between the Herpes Simplex Virus Type 1 Exonuclease, UL12, and the DNA Double-Strand Break-Sensing MRN Complex

ABSTRACT The herpes simplex virus type 1 (HSV-1) alkaline nuclease, encoded by the UL12 gene, plays an important role in HSV-1 replication, as a UL12 null mutant displays a severe growth defect. The HSV-1 alkaline exonuclease UL12 interacts with the viral single-stranded DNA binding protein ICP8 and promotes strand exchange in vitro in conjunction with ICP8. We proposed that UL12 and ICP8 form a two-subunit recombinase reminiscent of the phage lambda Red α/β recombination system and that the viral and cellular recombinases contribute to viral genome replication through a homologous recombination-dependent DNA replication mechanism. To test this hypothesis, we identified cellular interaction partners of UL12 by using coimmunoprecipitation. We report for the first time a specific interaction between UL12 and components of the cellular MRN complex, an important factor in the ATM-mediated homologous recombination repair (HRR) pathway. This interaction is detected early during infection and does not require viral DNA or other viral or cellular proteins. The region of UL12 responsible for the interaction has been mapped to the first 125 residues, and coimmunoprecipitation can be abolished by deletion of residues 100 to 126. These observations support the hypothesis that cellular and viral recombination factors work together to promote efficient HSV-1 growth.

Multiplexed RNA trafficking in oligodendrocytes and neurons

In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.

Single molecule imaging of translational output from individual RNA granules in neurons

Translation events in RNA granules in neurons are spatially clustered near individual granules, and translation output from individual granules is either sporadic or bursty. Translation of Venus-FMRP RNA is more bursty than translation of Venus-ARC RNA, and mGluR activation increases overall translation but decreases bursty translation.

A plasminogen-like protein selectively degrades stearoyl-CoA desaturase in liver microsomes

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum that is rapidly and selectively degraded when isolated liver microsomes are incubated at 37 °C. We previously reported the purification of a 90-kDa microsomal protein with SCD protease activity and characterized the inhibitor sensitivity of the protease. Here we show that the 90-kDa protein is a microsomal form of plasminogen (Pg) and that the purified SCD protease contains a spectrum of plasmin-like derivatives. The 90-kDa protein was identified as Pg by mass spectrometry of its tryptic peptides. The purified SCD protease reacted with Pg antibody, and immunoblotting demonstrated enrichment of Pg by the purification procedure established for the SCD protease. Analysis of microsomes by zymography demonstrated a single band of proteolytic activity at 70-kDa corresponding to the mobility of Pg in nonreduced polyacrylamide gels. When microsomes were incubated at 37 °C prior to zymography, an intense band of proteolytic activity developed at 30-kDa. The purified SCD protease displayed a spectrum of proteolytic bands ranging from 70 to 30 kDa. Degradation of SCD by the purified protease and by microsomes was inhibited by bdellin, a plasmin inhibitor from the medicinal leech Hirudo medicinalis. To explore the role of Pg in the degradation of SCD in vivo, we examined SCD expression and degradation in microsomes isolated from Pg-deficient (Pg–/–) mice. Compared with microsomes from wild-type littermate control mice, liver microsomes from Pg–/– mice had significantly higher levels of SCD. Degradation of SCD in microsomes from Pg–/– mice was markedly diminished, whereas liver microsomes from control mice showed rapid SCD degradation similar to that observed in rat liver microsomes. These findings indicate that SCD is degraded by a protease related to Pg and suggest that plasmin moonlights as an intracellular protease.

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ∼1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21–24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20–50 nucleotides was ∼3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.

Identification of New Proteins that Modulate the Germination of Spores of Bacillus Species

A number of operons encoding the nutrient germinant receptors (GRs) in dormant spores of Bacillus megaterium and Bacillus subtilis species have small open reading frames (ORFs) of unknown function within or immediately adjacent to the operons. Inactivation of the genes in these ORFs, encoding proteins now termed D proteins, either significantly increased or decreased spore germination via the associated GR but had no effects on germination via non-GR-dependent germinants. These effects on GR-dependent germination were complemented by ectopic expression of the appropriate D gene (gene encoding D protein). However, substitution of noncognate D genes in two GR operons resulted in inhibition of germination via the GR manipulated, although ectopic overexpression of a D gene had no effect on overall GR-dependent germination. The various D genes studied were expressed in the forespore during sporulation in parallel with the associated GR operon, and transcription of a B. subtilis D gene was controlled by RNA polymerase sigma factor σ(G). These results indicate that proteins encoded by small ORFs within or adjacent to operons encoding GRs play major roles in modulating GR function in spores of Bacillus species. In B. subtilis, deletion of a D gene (B. subtilis gerKD [gerKDbs]) adjacent to the gerK operon encoding the GerK GR or ectopic expression or overexpression of gerKDbs had no major effect on the levels of GR subunits or of two other germination proteins.