BackgroundNitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs.Methods/FindingsFifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer1177 was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr495 were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability.Conclusion/SignificanceThis human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.

Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.Ran | lysine acetylation | genetic code expansion concept | nucleus | nuclear cytosolic transport T he small GTP-binding protein Ran (Ras-related nuclear) is involved in nucleo-cytoplasmic transport processes, nuclear envelope formation, and the formation of the mitotic spindle (1). Ran, furthermore, has a variety of cytosolic functions and is involved in the cross-talk with the actin cytoskeleton. As a member of the Ras superfamily, Ran is structurally composed of a fold known as the G-domain (GTP-binding domain), a central sixstranded β-sheet that is surrounded by α-helices. Ras-family members bind to GTP and GDP nucleotides with high picomolar affinity. However, only in the GTP-bound form and the switch Iand switch II-loops adopt a stable conformation. Ran has been structurally characterized in great detail, including different nucleotide states and various protein complexes (2-4).In interphase cells, about 90% of cellular Ran is nuclear, and only a minor proportion is cytosolic (5). The localization of the guanine-nucleotide exchange factor (GEF) RCC1 (Regulator of chromosome condensation 1) at the nuclear chromatin and the RanGAP (RanGTPase-activating protein) at the cytosolic site of the nuclear pore creates a gradient of Ran•GTP in the nucleus and Ran•GDP in the cytosol (6-8).In the nucleus, Ran•GTP binds to exportins such as CRM1 (Chromosome region maintenance 1) to transport cargo proteins containing a nuclear export signal (NES) into the cytosol (3, 9, 10). Ran•GTP, furthermore, binds to Importin-β•cargo complexes to release the cargo in the nucleus (11-15). In the cytosol, the Importin•Ran•GTP complexes, as well as the ternary exportin•Ran•GTP-cargo complexes, dissociate on binding of RanBP1 and subsequent GTP hydrolysis catalyzed by RanGAP (16,17). The Ran transport cycle closes by translocation of Ran•GDP to the nucleus by the nuclear transport factor 2 (NTF2) (4,(17)(18)(19)(20). Many of these Ran interactions also ...

Rho proteins are small GTP/GDP-binding proteins primarily involved in cytoskeleton regulation. Their GTP/GDP cycle is often tightly connected to a membrane/cytosol cycle regulated by the Rho guanine nucleotide dissociation inhibitor ␣ (RhoGDI␣). RhoGDI␣ has been regarded as a housekeeping regulator essential to control homeostasis of Rho proteins. Recent proteomic screens showed that RhoGDI␣ is extensively lysine-acetylated. Here, we present the first comprehensive structural and mechanistic study to show how RhoGDI␣ function is regulated by lysine acetylation. We discover that lysine acetylation impairs Rho protein binding and increases guanine nucleotide exchange factor-catalyzed nucleotide exchange on RhoA, these two functions being prerequisites to constitute a bona fide GDI displacement factor. RhoGDI␣ acetylation interferes with Rho signaling, resulting in alteration of cellular filamentous actin. Finally, we discover that RhoGDI␣ is endogenously acetylated in mammalian cells, and we identify CBP, p300, and pCAF as RhoGDI␣-acetyltransferases and Sirt2 and HDAC6 as specific deacetylases, showing the biological significance of this post-translational modification.