Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

The insect neuropeptide prothoracicotropic hormone is released with a daily rhythm: re-evaluation of its role in development.

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two) critical moments early in each developmental stage at which time it triggers prolonged activation of the prothoracic glands to synthesize and release the steroid molting hormones (ecdysteroids), which elicit developmental responses in target tissues. We used an in vitro assay for PTTH released from excised brains of the bug Rhodnius prolixus and report that release of PTTH does occur at the expected time on day 6, but that this release is merely the first in a daily rhythm of release that continues throughout most of the 21 days of larval-adult development. This finding, together with reports of circadian control of ecdysteroid synthesis and titer throughout this time, raises significant challenges to several features of the current understanding of the hormonal control of insect development. New questions are raised concerning the function(s) of PTTH, its relationship with the prothoracic glands, and the significance of circadian rhythmicity throughout this endocrine axis. The significance of the reported observations derives from the set of entirely new questions they raise concerning the regulation of insect development.

Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target.

An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins. This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A. The U1A domain responsible for binding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage. Affinity selection identified four U1A residues in the mutagenized region that are important for specific binding to U1 hairpin II. One of these residues (Leu-49) disproportionately affects the rates of binding and release and appears to play a critical role in locking the protein onto the RNA. Interestingly, a protein variant that binds more tightly than U1A emerged during the selection, showing that the affinity of U1A for U1 RNA has not been optimized during evolution.

Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.

Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a potent inhibitory agent in all major forms of inflammation. To identify a potential mechanism of antiinflammatory action of alpha-MSH, we tested its effects on production of nitric oxide (NO), believed to be a mediator common to all forms of inflammation. We measured NO and alpha-MSH production in RAW 264.7 cultured murine macrophages stimulated with bacterial lipopolysaccharide and interferon gamma. alpha-MSH inhibited production of NO, as estimated from nitrite production and nitration of endogenous macrophage proteins. This occurred through inhibition of production of NO synthase II protein; steady-state NO synthase II mRNA abundance was also reduced. alpha-MSH increased cAMP accumulation in RAW cells, characteristic of alpha-MSH receptors in other cell types. RAW cells also expressed mRNA for the primary alpha-MSH receptor (melanocortin 1). mRNA for proopiomelanocortin, the precursor molecular of alpha-MSH, was expressed in RAW cells, and tumor necrosis factor alpha increased production and release of alpha-MSH. These results suggest that the proinflammatory cytokine tumor necrosis factor alpha can induce macrophages to increase production of alpha-MSH, which then becomes available to act upon melanocortin receptors on the same cells. Such stimulation of melanocortin receptors could modulate inflammation by inhibiting the production of NO. The results suggest that alpha-MSH is an autocrine factor in macrophages which modulates inflammation by counteracting the effects of proinflammatory cytokines.

Signal transduction in systemic acquired resistance.

Systemic acquired resistance (SAR) is an important component of plant defense against pathogen infection. Accumulation of salicylic acid (SA) is required for the induction of SAR. However, SA is apparently not the translocated signal but is involved in transducing the signal in target tissues. Interestingly, SA accumulation is not required for production and release of the systemic signal. In addition to playing a pivotal role in SAR signal transduction, SA is important in modulating plant susceptibility to pathogen infection and genetic resistance to disease. It has been proposed that SA inhibition of catalase results in H2O2 accumulation and that therefore H2O2 serves as a second messenger in SAR signaling. We find no accumulation of H2O2 in tissues expressing SAR; thus the role of H2O2 in SAR signaling is questionable.

Potent interleukin 3 receptor agonist with selectively enhanced hematopoietic activity relative to recombinant human interleukin 3.

A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10- to 20-fold greater biological activity than recombinant human IL-3 (rhIL-3) in human hematopoietic cell proliferation and marrow colony-forming-unit assays. In contrast, SC-55494 is only twice as active as rhIL-3 in priming the synthesis of inflammatory mediators such as leukotriene C4 and triggering the release of histamine from peripheral blood leukocytes. The enhanced hematopoietic activity of SC-55494 correlates with a 60-fold increase in IL-3 alpha-subunit binding affinity and a 20-fold greater affinity for binding to alpha/beta receptor complexes on intact cells relative to rhIL-3. SC-55494 demonstrates a 5- to 10-fold enhanced hematopoietic response relative to its ability to activate the priming and release of inflammatory mediators. Therefore, SC-55494 may ameliorate the myeloablation of cancer therapeutic regimens while minimizing dose-limiting inflammatory side effects.