Kidney collecting duct cell type composition is regulated by Notch signaling via modulation of mTORC1.

The plasticity and diversity of cell types with specialized functions likely defines the capacity of multicellular organisms to adapt to physiologic stressors. The kidney collecting ducts contribute to water, electrolyte, and pH homeostasis and are composed of mature intermingled epithelial cell types that are susceptible to transdifferentiate. The conversion of kidney collecting duct principal cells to intercalated cells is actively inhibited by Notch signaling to ensure urine concentrating capability. Here we identify Hes1, a target of Notch signaling, allows for maintenance of functionally distinct epithelial cell types within the same microenvironment by regulating mechanistic target of rapamycin complex 1 (mTORC1) activity. Hes1 directly represses the expression of insulin receptor substrate 1 ( Irs1 ), an upstream component of mTOR pathway and suppresses mTORC1 activity in principal cells. Genetic inactivation of tuberous sclerosis complex 2 ( Tsc2 ) to increase mTORC1 activity in mature principal cells is sufficient to promote acquisition of intercalated cell properties, while inhibition of mTORC1 in adult kidney epithelia suppresses intercalated cell properties. Considering that mTORC1 integrates environmental cues, the linkage of functionally distinct epithelial cell types to mTORC1 activity levels likely allows for cell plasticity to be regulated by physiologic and metabolic signals and the ability to sense/transduce these signals.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents.

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens. METHODS: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells. RESULTS: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP. CONCLUSIONS: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.

TAK1 Regulates the Nrf2 Antioxidant System Through Modulating p62/SQSTM1.

AIMS: Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is the master transcriptional regulator of antioxidant gene expression. On increased oxidative stress, an adaptor for Nrf2 degradation, Kelch-like ECH-associated protein 1 (Keap1), is directly modulated by oxidants in the cytoplasm, which results in stabilization and activation of Nrf2. Nrf2 is also constitutively active, to some extent, in the absence of exogenous oxidative stress. We have previously demonstrated that intestinal epithelium-specific TGF-ß-activated kinase 1 (TAK1) deletion downregulates the level of Nrf2 protein, resulting in an increase of reactive oxygen species (ROS) in a mouse model. We aim at determining the mechanism by which TAK1 modulates the level of Nrf2. RESULTS: We found that TAK1 upregulated serine 351 phosphorylation of an autophagic adaptor protein, p62/Sequestosome-1 (SQSTM1), which facilitates interaction between p62/SQSTM1 and Keap1 and subsequent Keap1 degradation. This, ultimately, causes increased Nrf2. Tak1 deficiency reduced the phosphorylation of p62/SQSTM1, resulting in decreased steady-state levels of Nrf2 along with increased Keap1. We also found that this regulation is independent of the canonical redox-mediated Nrf2 activation mechanism. In Tak1-deficient intestinal epithelium, a synthetic phenolic electrophile, butylated hydroxyanisole still effectively upregulated Nrf2 and reduced ROS. INNOVATION: Our results identify for the first time that TAK1 is a modulator of p62/SQSTM1-dependent Keap1 degradation and maintains the steady state-level of Nrf2. CONCLUSION: TAK1 regulates Nrf2 through modulation of Keap-p62/SQSTM1 interaction. This regulation is important for homeostatic antioxidant protection in the intestinal epithelium. Antioxid. Redox Signal. 25, 953-964.

Pseudorabies virus and herpes simplex virus type 1 utilize different tegument-glycoprotein interactions to mediate the process of envelopment.

BACKGROUND AND OBJECTIVE: During herpesvirus envelopment capsids, tegument polypeptides and membrane proteins assemble at the site of budding, and a cellular lipid bilayer becomes refashioned into a spherical envelope. A web of interactions between tegument proteins and the cytoplasmic tails of viral glycoproteins play a critical role in this process. We have previously demonstrated that for herpes simplex virus (HSV)-1 the cytoplasmic tail of glycoprotein H (gH) binds the tegument protein VP16. The HSV and pseudorabies virus (PRV) genomes are essentially collinear, and individual gene products show significant sequence homology. However, the demarcation of function often differs between PRV and HSV proteins. The goal of this study was to determine whether PRV gH and VP16 interact in a manner similar to their homologs in HSV. METHODS: A fusion protein pull-down assay was performed in which a PRV gH cytoplasmic tail-glutathione S-transferase fusion protein, bound to glutathione-Sepharose beads, was incubated with PRV-infected cell cytosol, washed and subjected to Western blot analysis using anti-PRV VP16 antisera. RESULTS: Western blots indicate that PRV VP16 does not specifically bind to the PRV gH tail. CONCLUSION: Our results highlight that, despite the relatively close evolutionary relationship between HSV and PRV, there are significant differences in their protein interactions that drive envelopment.

Recruitment of the oncoprotein v-ErbA to aggresomes.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.

Molecular discrimination of multiple primary versus metastatic squamous cell cancers of the head/neck and lung.

Patients with squamous cell carcinoma (SqCa) arising in the head and neck (H/N) commonly develop solitary pulmonary metastases that mimic the clinical, radiographic, and pathologic presentation of new primary lung SqCa. Primary pulmonary and metastatic SqCas cannot be differentiated from each other histologically. However, distinguishing multiple independent primary neoplasms from a primary H/N SqCa with pulmonary metastasis has prognostic significance due to its impact on tumor stage, the most important determinant of prognosis. Since genomic instability is a common feature of cancer, we hypothesized that independently-arising neoplasms in an individual patient would exhibit measurable genomic variation, enabling discrimination of tumor lineage and relatedness. In this study, we describe a molecular approach for analysis of genetic variation among multiple tumors from a single patient that does not rely on collection of normal tissue, and which can be performed with minimal tumor samples. Genomic DNA from H/N and lung SqCas from individual patients were analyzed by microsatellite PCR to identify discordant allelic variation. This method is rapid, sensitive, does not require constitutional DNA for comparison, and can be applied to the analysis of archival tumor DNA. Our results demonstrate that microsatellite PCR can identify discordant genetic variation among multiple tumors from a single patient, facilitating the molecular discrimination of metachronous primary SqCa versus solitary pulmonary metastasis from a H/N primary SqCa.