Binding of Dynein Light Chain (PIN) to Neuronal Nitric Oxide Synthase in the Absence of Inhibition

Rodrı́guez-Crespo, Ignacio; Straub, Wesley; Gavilanes, Francisco; Montellano, Paul R. Ortiz de

doi:10.1006/abbi.1998.0928

Cited by 56 publications

(49 citation statements)

References 39 publications

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“…However, the presence of competitive nNOS inhibitors such as 7-nitroindazole or L-N -nitroarginine-2,4-Ldiaminobutyric amide did not increase viral titers of r⌬LC8-N2c as with the levels of rN2c-infected cells (data not shown). Consistent with recent reports, our data also indicate that LC8 does not inhibit nNOS activity (44,45).…”

Section: Discussionsupporting

confidence: 93%

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

Tan¹,

Preuss²,

Williams

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

120

105

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Recent studies indicate that the interaction between rabies virus (RV) phosphoprotein and the dynein light chain 8 (LC8) is essential for RV pathogenesis. Through its association with the dynein motor complex, LC8 has been suggested as a molecular factor that links the viral ribonucleoprotein to the host cell transport system. Recent structural investigations, however, dispute this model. To understand the role of LC8 in RV pathogenesis, we generated recombinant RVs with or without the LC8 binding domain (LC8-BD) deleted from the RV phosphoprotein. Peripheral infection of adult mice showed that removal of the LC8-BD did not inhibit entry into the CNS, although it prevented onset of RV-induced CNS disease. However, deletion of the LC8-BD significantly attenuated viral transcription and replication in the CNS. Studies in RAG2 knockout (KO) mice infected with the same recombinant RVs confirmed this finding and indicated that the adaptive immune system is not a factor in the attenuation of viral replication early in the infection. In cell culture, the deletion of the LC8-BD greatly attenuated growth on neuronal cells whereas the growth pattern on nonneuronal cells remained unchanged. However, deletion of the LC8-BD did not affect production of RV virions. We provide evidence that removal of the LC8-BD decreases primary transcription. In this study, we propose that LC8 does not play a role in the retrograde axonal transport of RV and that the deletion of the LC8-BD impairs the infectivity of the virions by reducing early transcription and replication in neurons.pathogenesis ͉ negative-strand RNA viruses ͉ rhabdovirus ͉ viral transport R abies virus (RV) is a member of the family Rhabdoviridae that infects the CNS. RV has a relatively simple genome that encodes the five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix (M), glycoprotein (G), and RNA polymerase (L). Pathogenic RV infection without immediate postexposure prophylaxis frequently causes fatal encephalomyelitis in both humans and animals and has the distinction of having the highest case-fatality ratio among infectious pathogens (1). Past experiments in mice have suggested that street strains of RV initiate infection at the nerve terminals of the peripheral nervous system (PNS) and then travel along the axons located in the spinal cord (2, 3). Consequently, this axonal transport leads to RV infection of the brain (4). Yet, the molecular basis for the transport of the RV particle remains unclear and controversial. Most transneuronal studies of the CNS in nonhuman primates (5, 6) and mice (7,8) reveal that fixed (laboratory-adapted) strains of RV spread exclusively in the retrograde direction; however, past in vitro and in vivo studies suggest that anterograde transport might also be involved (7, 9-11). The discovery that the dynein light chain, LC8 (DNLC1), interacts with the RV phosphoprotein (RVP) provides a putative molecular link between the virus and host cell transport system and, thus, a potential mechanism for the neuroinvasive prope...

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Section: Discussionsupporting

confidence: 93%

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

Tan¹,

Preuss²,

Williams

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

120

105

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“…Furthermore, in vitro, a strong increase in PIN mRNA did not significantly affect nNOS catalytic activity, which contrasts with the initial identification of PIN as a specific inhibitor of nNOS catalytic activity (16). This could not be confirmed by others (38), but it must be emphasized that, at present, all studies have been performed using recombinant proteins and cells transfected with exogenous nNOS. Therefore, our finding of a slight nonsignificant reduction of nNOS activity after overexpression of endogenous PIN in ␤-cells expressing nNOS warrants further investigations to assess if PIN does regulate nNOS activity.…”

Section: Discussioncontrasting

confidence: 57%

Protein Inhibitor of Neuronal Nitric Oxide Synthase (PIN) Is a New Regulator of Glucose-Induced Insulin Secretion

et al. 2006

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We previously showed that pancreatic ␤-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting ␤-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucoseinduced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, N-nitro-L-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery. Diabetes 55: 3279 -3288, 2006 T he second messenger nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as NO synthases (NOSs). Three NOS isoforms have been identified, including the constitutive neuronal NO synthase (nNOS), endothelial NOS (eNOS), and the cytokine-inducible NOS (iNOS) (1). Constitutive NOSs produce small amounts of NO, implicated in physiological functions such as synaptic transmission (2) and vascular tone (3), whereas iNOS produces high amounts of NO, acting as a cytotoxic mediator in immune response (4). In addition to these beneficial roles, NO exerts deleterious effects as a result of its overproduction, leading to neuronal death during brain ischemia and certain neurodegenerative pathologies (5) and to autoimmune diseases (6). For these reasons, a tight regulation of NOS catalytic activity is essential for its physiological function. Modulation of iNOS expression by cytokines is believed to be the most important component of iNOS regulation, but posttranscriptional mechanisms also operate (7,8). As for the constitutive isoforms, catalytic activity is closely modulated by different mechanisms. The enzyme's activity is first controlled by the reversible binding of Ca 2ϩ -calmodulin (9), which allows the transfer of NADPH-derived electrons from the reductase to the oxygenase domain of the enzyme (10). It is also positively or negatively regulated through phosphorylation by various protein kinases such as cAMP-dependent protein kinase and protein kinase C (11,12). In addition to the role of intracellular Ca 2ϩ levels and protein kinases, constitutive NOSs are also regulated via protein-protein interactions. Thus, eNOS-associated protein-1 (ENAP-1...

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“…Moreover, DYNLL promotes dimerization as was observed with swallow, DIC, and myoVa (2,15,23,39). Hence, it is important to characterize dimer-to-dimer interactions of DYNLL complexes to get more insight into their biological role.…”

mentioning

confidence: 93%

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

Radnai

Rapali

Hódi

et al. 2010

Journal of Biological Chemistry

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LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K d values of 9 and 40 M, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy-and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K d values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K d value (3 M). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

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Binding of Dynein Light Chain (PIN) to Neuronal Nitric Oxide Synthase in the Absence of Inhibition

Cited by 56 publications

References 39 publications

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

Protein Inhibitor of Neuronal Nitric Oxide Synthase (PIN) Is a New Regulator of Glucose-Induced Insulin Secretion

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

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