Authors: Hellsberg E, Ecker GF, Stary-Weinzinger A, Forrest LR
Journal: PLoS One. 2019 Jun 28;14(6):e0217377. doi: 10.1371/journal.pone.0217377. eCollection 2019.
The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.
Authors: Cao X, Lu Y, Liu Y, Zhou Y, Song H, Zhang W, Davis D, Cui J, Hao S, Jung J, Wu Q, Park DM, Yang C
Journal: J Mol Med (Berl). 2019 Jun 14. doi: 10.1007/s00109-019-01802-z. [Epub ahead of print]
Chordoma, a malignant bone cancer, is highly resistant to conventional therapeutic approaches; this greatly limits radio- and chemotherapeutic options and disease management. In the present study, we investigated three patient-derived chordoma cell lines to elucidate the molecular mechanism of resistance to therapeutics. An in vitro high-throughput chemical screening assay and an in vivo xenograft model were used to identify novel chemosensitizers for chordoma. We found that patient-derived chordoma cell lines recapitulated disease phenotypes, which were highlighted by robust resistance to medical therapy manifested as lack of DNA damage accumulation. Mechanistically, the PARP DNA repair pathway was found to play a central role in this resistance. Chemical screening confirmed that PARP inhibitors could strikingly enhance temozolomide (TMZ) therapy in chordoma cells. Combining the FDA-approved PARP inhibitor, olaparib, with chemotherapeutics not only potentiated DNA damage accumulation, cell cycle arrest, and apoptosis in vitro but also suppressed chordoma xenograft expansion in vivo. We conclude that combining PARP inhibition with TMZ could be an effective therapeutic approach for the clinical management of chordoma.
KEY MESSAGES: The PARP DNA repair pathway enhances chemoresistance in chordoma cells. Combining PARP inhibitors with genotoxic agents induces chordoma cell cytotoxicity. PARP inhibitor combining with temozolomide suppresses growth of chordoma in vivo.
Authors: Landy R, Cheung LC, Berg CD, Chaturvedi AK, Robbins HA, Katki HA
Journal: Ann Intern Med. 2019 Jun 4. doi: 10.7326/M18-3617. [Epub ahead of print]
A new study by investigators from the National Cancer Institute used population data to model three different scenarios for selecting ever-smokers for lung cancer screening with low-dose CT. They determined that a new risk threshold could lower the ratio of individuals needed to screen in order to prevent one lung cancer death, improving on the performance of current United States Preventive Services Task Force (USPSTF) guidelines. The USPSTF is considering recommending externally validated risk assessment models for screening. Read more about the AIM study.
Authors: Zhou Z, Blandino P, Yuan Q, Shen PH, Hodgkinson CA, Virkkunen M, Watson SJ, Akil H, Goldman D
Journal: Proc Natl Acad Sci U S A. 2019 Jun 10. pii: 201820410. doi: 10.1073/pnas.1820410116. [Epub ahead of print]
Artificially selected model organisms can reveal hidden features of the genetic architecture of the complex disorders that they model. Addictions are disease phenotypes caused by different intermediate phenotypes and pathways and thereby are potentially highly polygenic. High responder (bHR) and low responder (bLR) rat lines have been selectively bred (b) for exploratory locomotion (EL), a behavioral phenotype correlated with novelty-seeking, impulsive response to reward, and vulnerability to addiction, and is inversely correlated with spontaneous anxiety and depression-like behaviors. The rapid response to selection indicates loci of large effect for EL. Using exome sequencing of HR and LR rats, we identified alleles in gene-coding regions that segregate between the two lines. Quantitative trait locus (QTL) analysis in F2 rats derived from a bHR × bLR intercross confirmed that these regions harbored genes affecting EL. The combined effects of the seven genome-wide significant QTLs accounted for approximately one-third of the total variance in EL, and two-thirds of the variance attributable to genetic factors, consistent with an oligogenic architecture of EL estimated both from the phenotypic distribution of F2 animals and rapid response to selection. Genetic association in humans linked APBA2, the ortholog of the gene at the center of the strongest QTL, with substance use disorders and related behavioral phenotypes. Our finding is also convergent with molecular and animal behavioral studies implicating Apba2 in locomotion. These results provide multilevel evidence for genes/loci influencing EL. They shed light on the genetic architecture of oligogenicity in animals artificially selected for a phenotype modeling a more complex disorder in humans.
Authors: Luby M, Hsia AW, Nadareishvili Z, Cullison K, Pednekar N, Adil MM, Latour LL
Journal: Stroke. 2019 Jun 26:STROKEAHA119025914. doi: 10.1161/STROKEAHA.119.025914. [Epub ahead of print]
Background and Purpose: The high prevalence of hyperintense acute reperfusion marker (HARM) seen after endovascular therapy is suggestive of blood-brain barrier disruption and hemorrhage risk and may be attributable to multiple thrombectomy passes needed to achieve recanalization.
Methods: Patients with acute stroke were included if they were screened from January 2015 through February 2019, received an acute ischemic stroke diagnosis involving the anterior circulation, treated with or without IV tPA (intravenous tissue-type plasminogen activator), consented to the NINDS Natural History Study, and imaged with a baseline magnetic resonance imaging before receiving endovascular therapy. Consensus image reads for HARM and hemorrhagic transformation were performed. Good clinical outcome was defined as 0-2 using the latest available modified Rankin Scale score.
Results: Eighty patients met all study criteria and were included in the analyses. Median age was 65 years, 64% female, 51% black/African American, median admit National Institutes of Health Stroke Scale=19, 56% treated with IV tPA, and 84% achieved Thrombolysis in Cerebral Infarction score of 2b/3. Multiple-pass patients had significantly higher rates of severe HARM at 24 hours (67% versus 29%; P=0.001), any hemorrhagic transformation (60% versus 36%; P=0.04) and poor clinical outcome (67% versus 36%; P=0.008). Only age (odds ratio, 1.1; 95% CI, 1.01-1.12; P=0.022) and severe HARM at 24 hours post-endovascular therapy were significantly associated with multiple passes (odds ratio, 7.2; 95% CI, 1.93-26.92; P=0.003).
Conclusions: In this exploratory study, multiple thrombectomy passes are independently associated with a significant increase in blood-brain barrier disruption detected at 24 hours. Patients with HARM post-endovascular therapy had a >7-fold increase in the odds of having multiple- versus single-pass thrombectomy.
Authors: Lin H, Rogers GT, Lunetta KL, Levy D, Miao X, Troy LM, Jacques PF, Murabito JM
Journal: Am J Clin Nutr. 2019 Jun 12. pii: nqz060. doi: 10.1093/ajcn/nqz060. [Epub ahead of print]
BACKGROUND: Genes in metabolic and nutrient signaling pathways play important roles in lifespan in model organisms and human longevity.
OBJECTIVE: The aim of this study was to examine the relation of a quantitative measure of healthy diet to gene expression in a community-based cohort.
METHODS: We used the 2015 Dietary Guidelines for Americans Adherence Index (DGAI) score to quantify key dietary recommendations of an overall healthy diet. Our current analyses included 2,220 Offspring participants (mean age 66 ± 9 y, 55.4% women) and 2941 Third-Generation participants (mean age 46 ± 9 y, 54.5% women) from the Framingham Heart Study. Gene expression was profiled in blood through the use of the Affymetrix Human Exon 1.0 ST Array. We conducted a transcriptome-wide association study of DGAI adjusting for age, sex, smoking, cell counts, and technical covariates. We also constructed a combined gene score from genes significantly associated with DGAI.
RESULTS: The DGAI was significantly associated with the expression of 19 genes (false discovery rate <0.05). The most significant gene, ARRDC3, is a member of the arrestin family of proteins, and evidence in animal models and human data suggests that this gene is a regulator of obesity and energy expenditure. The DGAI gene score was associated with body mass index (P = 1.4 × 10-50), fasting glucose concentration (P = 2.5 × 10-11), type 2 diabetes (P = 1.1 × 10-5), and metabolic syndrome (P = 1.8 × 10-32).
CONCLUSIONS: Healthier diet was associated with genes involved in metabolic function. Further work is needed to replicate our findings and investigate the relation of a healthy diet to altered gene regulation.
Authors: Skadow M, Penna VR, Galant-Swafford J, Shevach EM, Thornton AM
Journal: J Immunol. 2019 Jun 5. pii: ji1900388. doi: 10.4049/jimmunol.1900388. [Epub ahead of print]
The transcription factor Helios is expressed in a large percentage of Foxp3+ regulatory T (Treg) cells and is required for the maintenance of their suppressive phenotype, as mice with a selective deficiency of Helios in Treg cells spontaneously develop autoimmunity. However, mice with a deficiency of Helios in all T cells do not exhibit autoimmunity, despite the defect in the suppressor function of their Treg cell population, suggesting that Helios also functions in non-Treg cells. Although Helios is expressed in a small subset of CD4+Foxp3- and CD8+ T cells and its expression is upregulated upon T cell activation, its function in non-Treg cells remains unknown. To examine the function of Helios in CD4+Foxp3- T cells, we transferred Helios-sufficient or -deficient naive CD4+Foxp3- TCR transgenic T cells to normal recipients and examined their capacity to respond to their cognate Ag. Surprisingly, Helios-deficient CD4+ T cells expanded and differentiated into Th1 or Th2 cytokine-producing effectors in a manner similar to wild-type TCR transgenic CD4+ T cells. However, the primed Helios-deficient cells failed to expand upon secondary challenge with Ag. The tolerant state of the Helios-deficient memory T cells was not cell-intrinsic but was due to a small population of Helios-deficient naive T cells that had differentiated into Ag-specific peripheral Treg cells that suppressed the recall response in an Ag-specific manner. These findings demonstrate that Helios plays a role in the determination of CD4+ T cell fate.
Authors: Kumar S, Masison DC
Journal: PLoS Genet. 2019 Jun 26;15(6):e1008219. doi: 10.1371/journal.pgen.1008219. [Epub ahead of print]
Fes1 is a conserved armadillo repeat-containing Hsp70 nucleotide exchange factor important for growth at high temperature, proteasomal protein degradation and prion propagation. Depleting or mutating Fes1 induces a stress response and causes defects in these processes that are ascribed solely to disruption of Fes1 regulation of Hsp70. Here, we find Fes1 was essential for degradation of gluconeogenic enzymes by the vacuole import and degradation (Vid) pathway and for cell wall integrity (CWI), which is crucial for growth at high temperature. Unexpectedly, Fes1 mutants defective in physical or functional interaction with Hsp70 retained activities that support Vid and CWI. Fes1 and the Fes1 mutants bound to the Vid substrate Fbp1 in vitro and captured Slt2, a signaling kinase that regulates CWI, from cell lysates. Our data show that the armadillo domain of Fes1 binds proteins other than Hsp70, that Fes1 has important Hsp70-independent roles in the cell, and that major growth defects caused by depleting Fes1 are due to loss of these functions rather than to loss of Hsp70 regulation. We uncovered diverse functions of Fes1 beyond its defined role in regulating Hsp70, which points to possible multi-functionality among its conserved counterparts in other organisms or organelles.
Authors: Balasubramanian A, Markovski M, Hoskins JR, Doyle SM, Wickner S
Journal: Proc Natl Acad Sci U S A. 2019 Jun 3. pii: 201904014. doi: 10.1073/pnas.1904014116. [Epub ahead of print]
Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec. Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ-Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.
Authors: Larsen BM, Cowan JE, Wang Y, Tanaka Y, Zhao Y, Voisin B, Constantinides MG, Nagao K, Belkaid Y, Awasthi P, Takahama Y, Bhandoola A
Journal: J Immunol. 2019 Jun 26. pii: ji1801540. doi: 10.4049/jimmunol.1801540. [Epub ahead of print]
The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1. Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.