Birt-Hogg-Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1͞2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5 AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt-Hogg-Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and͞or nutrient sensing through the AMPK and mTOR signaling pathways.hamartoma syndrome ͉ renal cancer ͉ Birt-Hogg-Dubé ͉ tumor suppressor B irt-Hogg-Dubé (BHD) syndrome predisposes patients to develop hair follicle hamartomas, lung cysts, and an increased risk for renal neoplasia (1-3). BHD patients develop bilateral, multifocal renal tumors with a variety of histologies (4). We mapped the BHD locus to chromosome 17p11.2 by linkage analysis in BHD kindreds (5, 6) and identified germ-line mutations in a gene with unknown function that is highly conserved (7,8). Twenty-two unique mutations predicted to truncate the BHD protein folliculin (FLCN), including a ''hot spot'' insertion͞deletion in a C 8 tract, were identified in 84% of BHD kindreds (9). Somatic ''second-hit'' mutations identified in BHD-associated renal tumors suggest a tumor-suppressor function for FLCN (10), underscored by loss of BHD mRNA expression in renal tumors from BHD patients (11).Recent studies suggest that several hamartoma syndromes may be linked through the convergent energy͞nutrient-sensing pathways involved in mammalian target of rapamycin (mTOR) regulation (12-15). These inherited syndromes are characterized by multiple hamartomas and an increased risk of cancer. Germ-line mutations have been identified in four causative genes: LKB1, responsible for Peutz-Jeghers syndrome (16-18), TSC1 and TSC2, responsible for tuberous sclerosis complex (TSC) (19), and PTEN, responsible for Cowden syndrome (20). Loss of gene function leads to dysregulation of mTOR, which regulates cell growth and size through stimulation of protein synthesis (15, 21, 22).BHD syndrome, also a hamartoma disorder, displays phenotypic similarities to TSC that have led to speculation that BHD may function in the pathway(s) signaling through mTOR (12,23). To ascertain FLCN function,...

SummaryThe promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely de®ned because of its degeneracy and asymmetry and seemingly variable location with respect to the À10 and À35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more de®ned marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the À35 hexamer are oriented opposite those closer to the À35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9,089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the US population (n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants (n = 11,382) provided medical, geographic, demographic, and socioeconomic information, and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. The majority (88.7%) of blood samples were collected between May 10th and July 31st, 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI: 2.6-6.5%) with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%; the highest seropositivity estimates were in African American participants, younger, female, and Hispanic participants, and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States.

Summary Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator–DNA–RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator–DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA–DNA–RNP and SoxS–DNA–RNP complexes are more stable than the corresponding Rob–DNA–RNP complex, although the binary Rob–DNA complex is often more stable than the corresponding MarA– or SoxS–DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/ Rob system. We suggest that activator–RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.