Novel Regulation of Type IV Collagenase (Matrix Metalloproteinase-9 and -2) Activities by Transforming Growth Factor-β1 in Human Prostate Cancer Cell Lines

The type IV collagenases/gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9 play a variety of important roles in both physiological and pathological processes and are regulated by various growth factors, including transforming growth factor-β1 (TGF-β1), in several cell types. Previous studies have suggested that cellular control of one or both collagenases can occur through direct transcriptional mechanisms and/or after secretion through proenzyme processing and interactions with metalloproteinase inhibitors. Using human prostate cancer cell lines, we have found that TGF-β1 induces the MMP-9 proenzyme; however, this induction does not result from direct effects on gene transcription but, instead, through a protein synthesis–requiring process leading to increased MMP-9 mRNA stability. In addition, we have examined levels of TGF-β1 regulation of MMP-2 in one prostate cancer cell line and found that TGF-β1 induces higher secreted levels of this collagenase through increased stability of the secreted 72-kDa proenzyme. These results identify two novel nontranscriptional pathways for the cellular regulation of MMP-9 and MMP-2 collagenase gene expression and activities.

Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non–calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

Direct observation of extension and retraction of type IV pili

Type IV pili are thin filaments that extend from the poles of a diverse group of bacteria, enabling them to move at speeds of a few tenths of a micrometer per second. They are required for twitching motility, e.g., in Pseudomonas aeruginosa and Neisseria gonorrhoeae, and for social gliding motility in Myxococcus xanthus. Here we report direct observation of extension and retraction of type IV pili in P. aeruginosa. Cells without flagellar filaments were labeled with an amino-specific Cy3 fluorescent dye and were visualized on a quartz slide by total internal reflection microscopy. When pili were attached to a cell and their distal ends were free, they extended or retracted at rates of about 0.5 μm s−1 (29°C). They also flexed by Brownian motion, exhibiting a persistence length of about 5 μm. Frequently, the distal tip of a filament adsorbed to the substratum and the filament was pulled taut. From the absence of lateral deflections of such filaments, we estimate tensions of at least 10 pN. Occasionally, cell bodies came free and were pulled forward by pilus retraction. Thus, type IV pili are linear actuators that extend, attach at their distal tips, exert substantial force, and retract.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics

Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-ψ[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+ channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type α1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α1A subunit. The resulting chimeric α1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type α1C subunit and 92% of the binding energy of the l-type α1S subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca2+ channels containing α1A/DHPS was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of α1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Topoisomerase IV is a target of quinolones in Escherichia coli.

We have demonstrated that, in Escherichia coli, quinolone antimicrobial agents target topoisomerase IV (topo IV). The inhibition of topo IV becomes apparent only when gyrase is mutated to quinolone resistance. In such mutants, these antibiotics caused accumulation of replication catenanes, which is diagnostic of a loss of topo IV activity. Mutant forms of topo IV provided an additional 10-fold resistance to quinolones and prevented drug-induced catenane accumulation. Drug inhibition of topo IV differs from that of gyrase. (i) Wild-type topo IV is not dominant over the resistant allele. (ii) Inhibition of topo IV leads to only a slow stop in replication. (iii) Inhibition of topo IV is primarily bacteriostatic. These differences may result from topo IV acting behind the replication fork, allowing for repair of drug-induced lesions. We suggest that this and a slightly higher intrinsic resistance of topo IV make it secondary to gyrase as a quinolone target. Our results imply that the quinolone binding pockets of gyrase and topo IV are similar and that substantial levels of drug resistance require mutations in both enzymes.