Review discusses evolutionary conserved functions of extracellular ubiquitin as a control mechanism to balance and/or prevent exuberant inflammation.
Ubiquitin is a post‐translational protein ...modifier and plays essential roles in all aspects of biology. Although the discovery of ubiquitin introduced this highly conserved protein as a molecule with extracellular actions, the identification of ubiquitin as the ATP‐dependent proteolysis factor 1 has focused subsequent research on its important intracellular functions. Little attention has since been paid to its role outside of the cell. During recent years, multiple observations suggest that extracellular ubiquitin can modulate immune responses and that exogenous ubiquitin has therapeutic potential to attenuate exuberant inflammation and organ injury. These observations have not been integrated into a comprehensive assessment of its possible role as an endogenous immune modulator. This review recapitulates the current knowledge about extracellular ubiquitin and discusses an emerging facet of its role in biology during infectious and noninfectious inflammation. The synopsis of these data along with the recent identification of ubiquitin as a CXCR4 agonist suggest that extracellular ubiquitin may have pleiotropic roles in the immune system and functions as an endogenous opponent of DAMPs. Functions of extracellular ubiquitin could constitute an evolutionary conserved control mechanism aimed to balance the immune response and prevent exuberant inflammation. Further characterization of its mechanism of action and cellular signaling pathways is expected to provide novel insights into the regulation of the innate immune response and opportunities for therapeutic interventions.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Ubiquitin is one of the most highly conserved proteins in eukaryotes and plays major biological roles as a post-translational protein modifier. Ubiquitin is also a natural constituent of plasma, and ...several lines of evidence suggest that extracellular ubiquitin is an immune modulator with anti-inflammatory properties. In addition, ubiquitin treatment has been shown to limit inflammation and reduce organ injury in various disease models and species in vivo. However, its mechanism of action is unknown. Here we show that extracellular ubiquitin is a natural CXC chemokine receptor 4 (CXCR4 and CD184) agonist. Extracellular ubiquitin promotes intracellular Ca2+ flux and reduces cAMP levels through a G protein-coupled receptor that signals via a Gαi/o protein in THP1 cells. Toll-like receptor 4 stimulation reduces ubiquitin-binding sites, which enabled identification of four Gαi/o PCRs as ubiquitin receptor candidates. Overexpression of candidate genes in HEK293 cells, gene silencing in THP1 cells, competition binding, and signaling studies with the CXCR4 agonist stromal cell-derived factor-1α (chemokine (CXC motif) ligand 12) and inhibitor AMD3100 identify CXCR4 as a functional ubiquitin receptor. Our finding uncovers a fundamentally new aspect of the role of ubiquitin in biology, has implications for the understanding of CXCR4-mediated events, and is expected to facilitate development of new therapeutic avenues for a variety of diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with alpha.sub.1A/B/D -adrenoceptors (ARs) and arginine vasopressin ...receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed alpha.sub.1A/B/D -ARs and detected that AVPR1A also heteromerizes with alpha.sub.1A/B/D -ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among alpha.sub.1 -AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and alpha.sub.1A/B/D -ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, alpha.sub.1a -AR and AVPR1A form hetero-oligomeric complexes composed of 2-4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Evidence suggests that ethanol-induced hypertension is associated with increased cardiovascular responsiveness to vasopressors in vivo and enhanced reactivity of isolated arteries to vasopressors ex ...vivo. The underlying mechanisms are not well understood and the contribution of ethanol metabolites to vascular effects induced by ethanol consumption are unclear. Mesenteric resistance arteries were harvested from Sprague-Dawley rats. Pressure myography was utilized to test effects of ethanol, acetaldehyde and phosphatidylethanol on myogenic tone and on vasoconstriction induced by phenylephrine, arginine vasopressin (aVP), endothelin-1 and KCl. Ethanol, acetaldehyde and phosphatidylethanol concentrations were monitored during the experiments. Ethanol concentrations in the vessel bath decreased with a half-life of 25min; acetaldehyde and phosphatidylethanol concentrations remained constant. Pretreatment with ethanol dose-dependently increased the potency of phenylephrine to induce vasoconstriction 4-fold (p<0.01). These effects were comparable when arteries were pre-treated with a single dose of ethanol for 30min and when ethanol concentrations were kept constant during 30min and 60min of pretreatment. While ethanol also dose-dependently increased the potency of aVP to induce vasoconstriction 1.7-fold (p<0.05), it did not affect vasoconstriction induced by endothelin-1 or KCl. Acetaldehyde pre-treatment (30 min) dose-dependently increased the potency of phenylephrine to induce vasoconstriction 2.7-fold (p<0.01) but did not affect other vasoconstrictor responses. Phosphatidylethanol did not affect any vasoconstrictor responses. Ethanol and its metabolites did not affect myogenic tone. These data suggest that ethanol and acetaldehyde selectively sensitize intrinsic constrictor responses upon activation of vascular α1-adrenergic and/or vasopressin receptors at clinically relevant concentrations. Our findings support the concept that enhanced vasoreactivity to vasoactive hormones contributes to the development of hypertension induced by ethanol consumption. Ex vivo exposure of resistance arteries to ethanol and acetaldehyde resembles effects of chronic ethanol consumption on intrinsic vascular function, and thus could serve as test platform to evaluate interventions aimed to mitigate vascular effects associated with ethanol consumption.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of ...the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of ...AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and β-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not β-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit β-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
We reported previously that α1-adrenoceptor (α1-AR) ligands inhibit chemokine receptor (CR) heteromerization partners of α1B/D-AR. The underlying mechanisms are unknown and in vivo evidence for such ...effects is missing. Utilizing CCR2 and α1B-AR as prototypical partners, we observed in recombinant systems and THP-1 cells that α1B-AR enhanced whereas its absence inhibited Gαi signaling of CCR2. Phenylephrine and phentolamine reduced the CCR2:α1B-AR heteromerization propensity and inhibited Gαi signaling of CCR2. Phenylephrine cross-recruited β-arrestin-2 to CCR2, and reduced expression of α1B/D-AR, CR partners (CCR1/2, CXCR4) and corresponding heteromers. Phentolamine reduced CR:α1B/D-AR heteromers without affecting β-arrestin-2 recruitment or receptor expression. Phenylephrine/phentolamine prevented leukocyte infiltration mediated via CR heteromerization partners in a murine air pouch model. Our findings document that α1-AR ligands inhibit leukocyte migration mediated by CR heteromerization partners in vivo and suggest interference with α1B-AR:CR heteromerization as a mechanism by which CR partners are inhibited. These findings provide new insights into the pharmacology of GPCR heteromers and indicate that an agonist and antagonist at one GPCR can act as antagonists at heteromerization partners of their target receptors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Systemic concentrations of chemokine CCL2, an agonist at chemokine receptors CCR2/3/5, have been associated with hemodynamic instability after traumatic-hemorrhagic shock. We reported previously that ...the CCR2 antagonist INCB3284 prevents cardiovascular collapse and reduces fluid requirements after 30min of hemorrhagic shock (HS), whereas the CCR5 antagonist Maraviroc was ineffective. The effects of CCR3 blockade after HS are unknown and information on the therapeutic potential of INCB3284 after longer periods of HS and in HS models in the absence of fluid resuscitation (FR) is lacking. The aims of the present study were to assess the effects of CCR3 blockade with SB328437 and to further define the therapeutic efficacy of INCB3284. In series 1-3, Sprague-Dawley rats were hemorrhaged to a mean arterial blood pressure (MAP) of 30mmHg, followed by FR to MAP of 60mmHg or systolic blood pressure of 90mmHg. Series 1: 30min HS and FR until t = 90min. SB328437 at t = 30min dose-dependently reduced fluid requirements by >60%. Series 2: 60min HS and FR until t = 300min. INCB3284 and SB328437 at t = 60min reduced fluid requirements by more than 65% (p<0.05 vs. vehicle) and 25% (p>0.05 vs. vehicle), respectively, until t = 220min. Thereafter, all animals developed a steep increase in fluid requirements. Median survival time was 290min with SB328437 and >300min after vehicle and INCB3284 treatment (p<0.05). Series 3: HS/FR as in series 2. INCB3284 at t = 60min and t = 200min reduced fluid requirements by 75% until t = 300min (p<0.05 vs. vehicle). Mortality was 70% with vehicle and zero with INCB3284 treatment (p<0.05). Series 4: INCB3284 and SB328437 did not affect survival time in a lethal HS model without FR. Our findings further support the assumption that blockade of the major CCL2 receptor CCR2 is a promising approach to improve FR after HS and document that the dosing of INCB3284 can be optimized.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We compared therapeutic properties of natural and engineered chemokine (C-X-C motif) receptor 4 (CXCR4) agonists in a rat acute respiratory distress syndrome (ARDS) model utilizing the PaO
/FiO
...-ratio as a clinically relevant primary outcome criterion. Ventilated rats underwent unilateral lung ischemia from t = 0-70 min plus hemorrhage to a mean arterial blood pressure (MAP) of 30 mmHg from t = 40-70 min, followed by reperfusion/fluid resuscitation until t = 300 min. Natural CXCR4 agonists (CXCL12, ubiquitin) and engineered CXCL12 variants (CXCL12
, CXCL2
, CXCL12K27A/R41A/R47A, CXCL12 (3-68)) were administered within 5 min of fluid resuscitation. Animals treated with vehicle or CXCL12 (3-68) reached criteria for mild and moderate ARDS between t = 90-120 min and t = 120-180 min, respectively, and remained in moderate ARDS until t = 300 min. Ubiquitin, CXCL12, CXCL12
and CXCL12
prevented ARDS development. Potencies of CXCL12/CXCL12
/CXCL12
were higher than the potency of ubiquitin. CXCL12K27A/R41A/R47A was inefficacious. CXCL12
> CXCL12 stabilized MAP and reduced fluid requirements. CXCR4 agonists at doses that preserved lung function reduced histological injury of the post-ischemic lung and reduced mortality from 55 to 9%. Our findings suggest that CXCR4 protein agonists prevent development of ARDS and reduce mortality in a rat model, and that development of new engineered protein therapeutics with improved pharmacological properties for ARDS is possible.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
It is unknown whether heteromerization between chemokine (C‐X‐C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b‐adrenoceptor (α1b‐AR) influences effects of the CXCR4/ACKR3 ...agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist‐promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4‐mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit β‐arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero‐trimerize with α1b‐AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from hetero‐oligomers comprising CXCR4. CXCL12 enhances phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from CXCR4:α1b‐AR heterodimers and reduces phenylephrine‐stimulated α1b‐AR‐promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand‐dependent functions of the receptor partners.
We provide biophysical evidence that chemokine (C‐X‐C motif) ligand 12 (CXCL12) and ubiquitin stimulate chemokine (C‐X‐C motif) receptor 4 (CXCR4)‐mediated Gαi activation, but that ubiquitin fails to recruit β‐arrestin to CXCR4. Both ligands differentially modulate the conformation of CXCR4, atypical chemokine receptor 3 (ACKR3) and α1b‐adrenoceptor hetero‐oligomers. Ubiquitin enhances α1b‐adrenoceptor‐promoted Gαq activation from hetero‐oligomers comprising CXCR4. CXCL12 enhances α1b‐AR‐promoted Gαq activation from CXCR4:α1b‐AR heterodimers and reduces α1b‐AR‐promoted Gαq activation from ACKR3 comprising hetero‐oligomers.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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