Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways

Epithelial Na⁺ absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na⁺ channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.     

Elevated levels of soluble TNF receptors 1 and 2 correlate with Plasmodium falciparum parasitemia in pregnant women: potential markers for malaria-associated inflammation

Plasmodium falciparum-infected erythrocytes (IEs) sequester in the intervillous space (IVS) of the placenta causing placental malaria (PM), a condition that increases a woman's chances of having a low-birth-weight baby. Because IEs sequester, they frequently are not observed in peripheral blood smears, resulting in women with PM being misdiagnosed and thus not treated. Because sequestered IEs induce inflammation in the IVS, detection of inflammatory mediators in the peripheral blood may provide an approach for diagnosing PM. Two counterregulatory molecules, TNF-αR (TNFR) 1 and TNFR2, modulate the pathological effects of TNF-α. Levels of these soluble TNFRs (sTNFRs) are reported to be elevated in children with severe malaria, but it is unclear if they are increased in the peripheral blood of PM-positive women with asymptomatic infections. In this study, sTNFR levels were measured throughout the course of pregnancy, as well as at delivery, in women with asymptomatic infections and those who remained uninfected. Results showed that both sTNFRs were significantly increased in the peripheral blood of women with asymptomatic malaria (p < 0.0001) and were positively correlated with parasitemia (p < 0.0001 for sTNFR1 and p = 0.0046 for sTNFR2). Importantly, levels of sTNFR2 were elevated in the peripheral blood of women who were PM-positive but peripheral blood-smear negative (p = 0.0017). Additionally, sTNFR2 levels were elevated in the blood of malaria-positive women who delivered low-birth-weight babies. In vitro studies demonstrated that syncytiotrophoblasts were not a major source of sTNFR. These data suggest that sTNFR2 may be a valuable biomarker for detection of malaria-associated inflammation.     

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1

Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H⁺ efflux by a Na⁺-H⁺ exchanger (NHE; Ddnhe1⁻), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1⁻ cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1⁻ cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H⁺ efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1⁻ cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1⁻ cells is determined primarily by loss of cofilin-dependent actin dynamics.     

Acute Administration of Non-Classical Estrogen Receptor Agonists Attenuates Ischemia-Induced Hippocampal Neuron Loss in Middle-Aged Female Rats

Background

Pretreatment with 17β-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown.

Methodology/Principal Findings

We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 µg), G1 (50 µg) or STX (50 µg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 µg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55–60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices.

Conclusions/Significance

These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.     

Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality

Background

Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8⁺ T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8⁺ T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.

Methodology/Principal Findings

Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8⁺ T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8⁺ T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8⁺ T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8⁺ T-cells.

Conclusions

These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8⁺ T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680 [{"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680]     

The Parkinson's Disease Associated LRRK2 Exhibits Weaker In Vitro Phosphorylation of 4E-BP Compared to Autophosphorylation

Mutations in the gene encoding Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson''s disease (PD). LRRK2 is a multi-domain protein kinase containing a central catalytic core and a number of protein-protein interaction domains. An important step forward in the understanding of both the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological substrates. In the present study we examined phosphorylation of 4E-BP (eukaryotic initiation factor 4E (eIF4E)-binding protein), a recently proposed substrate for LRRKs. We found that LRRK2 is capable of phosphorylating 4E-BP in vitro. The PD related LRRK2-G2019S mutant was ∼2 fold more active than wild type protein. However, LRRK2 autophosphorylation was stronger than 4E-BP phosphorylation under conditions of molar excess of 4E-BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E-BP at the same site as LRRK2 in vitro. Finally, we did not see changes in 4E-BP phosphorylation levels using inducible expression of LRRK2 in HEK cell lines. We also found that MAPK14/p38α phosphorylates 4E-BP in transient overexpression experiments whereas LRRK2 did not. We suggest that increased 4E-BP phosphorylation reported in some systems may be related to p38-mediated cell stress rather than direct LRRK2 activity. Overall, our results suggest that 4E-BP is a relatively poor direct substrate for LRRK2.