Authors: Jeon J, Thurber KR, Ghirlando R, Yau WM, Tycko R
Journal: Proc Natl Acad Sci U S A. 2019 Aug 6. pii: 201908006. doi: 10.1073/pnas.1908006116. [Epub ahead of print]
Common experimental approaches for characterizing structural conversion processes such as protein folding and self-assembly do not report on all aspects of the evolution from an initial state to the final state. Here, we demonstrate an approach that is based on rapid mixing, freeze-trapping, and low-temperature solid-state NMR (ssNMR) with signal enhancements from dynamic nuclear polarization (DNP). Experiments on the folding and tetramerization of the 26-residue peptide melittin following a rapid pH jump show that multiple aspects of molecular structure can be followed with millisecond time resolution, including secondary structure at specific isotopically labeled sites, intramolecular and intermolecular contacts between specific pairs of labeled residues, and overall structural order. DNP-enhanced ssNMR data reveal that conversion of conformationally disordered melittin monomers at low pH to α-helical conformations at neutral pH occurs on nearly the same timescale as formation of antiparallel melittin dimers, about 6 to 9 ms for 0.3 mM melittin at 24 °C in aqueous solution containing 20% (vol/vol) glycerol and 75 mM sodium phosphate. Although stopped-flow fluorescence data suggest that melittin tetramers form quickly after dimerization, ssNMR spectra show that full structural order within melittin tetramers develops more slowly, in ∼60 ms. Time-resolved ssNMR is likely to find many applications to biomolecular structural conversion processes, including early stages of amyloid formation, viral capsid formation, and protein-protein recognition.
Authors: Deuse T, Hu X, Agbor-Enoh S, Koch M, Spitzer MH, Gravina A, Alawi M, Marishta A, Peters B, Kosaloglu-Yalcin Z, Yang Y, Rajalingam R, Wang D, Nashan B, Kiefmann R, Reichenspurner H, Valantine H, Weissman IL, Schrepfer S
Journal: Nat Biotechnol. 2019 Aug 19. doi: 10.1038/s41587-019-0227-7. [Epub ahead of print]
The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.
Authors: Hamaneh MB, Yu YK
Journal: PLoS One. 2019 Aug 2;14(8):e0220742. doi: 10.1371/journal.pone.0220742. eCollection 2019.
Reprogramming of somatic cells to induced pluripotent stem cells, by overexpressing certain factors referred to as the reprogramming factors, can revolutionize regenerative medicine. To provide a coherent description of induced pluripotency from the gene regulation perspective, we use 35 microarray datasets to construct a reprogramming gene regulatory network. Comprising 276 nodes and 4471 links, the resulting network is, to the best of our knowledge, the largest gene regulatory network constructed for human fibroblast reprogramming and it is the only one built using a large number of experimental datasets. To build the network, a model that relates the expression profiles of the initial (fibroblast) and final (induced pluripotent stem cell) states is proposed and the model parameters (link strengths) are fitted using the experimental data. Twenty nine additional experimental datasets are collectively used to test the model/network, and good agreement between experimental and predicted gene expression profiles is found. We show that the model in conjunction with the constructed network can make useful predictions. For example, we demonstrate that our approach can incorporate the effect of reprogramming factor stoichiometry and that its predictions are consistent with the experimentally observed trends in reprogramming efficiency when the stoichiometric ratios vary. Using our model/network, we also suggest new (not used in training of the model) candidate sets of reprogramming factors, many of which have already been experimentally verified. These results suggest our model/network can potentially be used in devising new recipes for induced pluripotency with higher efficiencies. Additionally, we classify the links of the network into three classes of different importance, prioritizing them for experimental verification. We show that many of the links in the top ranked class are experimentally known to be important in reprogramming. Finally, comparing with other methods, we show that using our model is advantageous.
Authors: Coghill AE, Suneja G, Rositch AF, Shiels MS, Engels EA
Journal: JAMA Oncol. 2019 Aug 1. doi: 10.1001/jamaoncol.2019.1742. [Epub ahead of print]
IMPORTANCE: HIV-infected patients with cancer have an elevated cancer-specific mortality rate compared with HIV-uninfected patients with cancer. However, to our knowledge, studies describing this association have not adjusted in detail for cancer treatment, despite evidence of suboptimal cancer treatment in the setting of HIV.
OBJECTIVE: To compare cancer-specific mortality in HIV-infected and HIV-uninfected patients with cancer after adjusting for available data on receipt of specific cancer treatments.
DESIGN, SETTING, PARTICIPANTS: We used Surveillance, Epidemiology, and End Results-Medicare linked data to identify 308,268 patients in the United States (age, ≥65 years), including 288 with HIV infection, with nonadvanced cancers of the colorectum, lung, prostate, or breast diagnosed between 1996 and 2012 who received standard, stage-appropriate cancer treatment during the year after cancer diagnosis. Data analysis was done from August 2016 to September 2018.
EXPOSURES: HIV infection identified by the presence of Medicare claims.
MAIN OUTCOMES: Overall mortality, cancer-specific mortality, and relapse or cancer-specific mortality after initial treatment.
RESULTS: In this database study of 308,268 patients with nonadvanced cancer (168,998 men and 139,270 women; age, ≥65 years), HIV-infected patients (n = 288) had significant elevations in the overall mortality rate compared with HIV-uninfected patients for cancers of the colorectum (hazard ratio [HR], 1.73; 95% CI, 1.11-2.68; P = .02), prostate (HR, 1.58; 95% CI, 1.23-2.03; P < .01), and breast (HR, 1.50; 95% CI, 1.01-2.24; P = .05). Cancer-specific mortality was elevated for prostate (HR, 1.65; 95% CI, 0.98-2.79; P = .06) and breast cancer (HR, 1.85; 95% CI, 0.96-3.55; P = .07). Compared with their HIV-uninfected counterparts, HIV-infected men with prostate cancer also experienced significantly higher rates of relapse or death (HR, 1.32; 95% CI, 1.03-1.71; P = .03) as did HIV-infected women with breast cancer (HR, 1.63; 95% CI, 1.09-2.43; P = .02).
CONCLUSIONS AND RELEVANCE: In the United States, elderly HIV-infected patients with cancer, particularly prostate and breast cancers, have worse outcomes than HIV-uninfected patients with cancer. This disparity persists even after adjustment for administered first-course cancer treatments and will become increasingly relevant as the HIV population in the United States continues to age.
Authors: Han TU, Root J, Reyes LD, Huchinson EB, Hoffmann JD, Lee WS, Barnes TD, Drayna D
Journal: Proc Natl Acad Sci U S A. 2019 Aug 12. pii: 201901480. doi: 10.1073/pnas.1901480116. [Epub ahead of print]
Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and β (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.
Authors: Puri R, Cheng XT, Lin MY, Huang N, Sheng ZH
Journal: Nat Commun. 2019 Aug 13;10(1):3645. doi: 10.1038/s41467-019-11636-5.
Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Here, we reveal Mul1-Mfn2 pathway that maintains neuronal mitochondrial integrity under stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mito tethering antagonist. Reduced ER-Mito coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mito anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mito contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction and altered ER-Mito interplay.
Authors: Lingemann M, McCarty T, Liu X, Buchholz UJ, Surman S, Martin SE, Collins PL, Munir S
Journal: PLoS Pathog. 2019 Aug 5;15(8):e1007963. doi: 10.1371/journal.ppat.1007963. [Epub ahead of print]
Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.
Authors: Christenson ET, Isaac DT, Yoshida K, Lipo E, Kim JS, Ghirlando R, Isberg RR, Banerjee A
Journal: Proc Natl Acad Sci U S A. 2019 Aug 20. pii: 201902806. doi: 10.1073/pnas.1902806116. [Epub ahead of print]
Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+. Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.
Authors: Chen YC, Elnitski L
Journal: PLoS Comput Biol. 2019 Jul 22;15(7):e1007095. doi: 10.1371/journal.pcbi.1007095. [Epub ahead of print]
Alternative transcript isoforms are common in tumors and act as potential drivers of cancer. Mechanisms determining altered isoform expression include somatic mutations in splice regulatory sites or altered splicing factors. However, since DNA methylation is known to regulate transcriptional isoform activity in normal cells, we predicted the highly dysregulated patterns of DNA methylation present in cancer also affect isoform activity. We analyzed DNA methylation and RNA-seq isoform data from 18 human cancer types and found frequent correlations specifically within 11 cancer types. Examining the top 25% of variable methylation sites revealed that the location of the methylated CpG site in a gene determined which isoform was used. In addition, the correlated methylation-isoform patterns classified tumors into known subtypes and predicted distinct protein functions between tumor subtypes. Finally, methylation-correlated isoforms were enriched for oncogenes, tumor suppressors, and cancer-related pathways. These findings provide new insights into the functional impact of dysregulated DNA methylation in cancer and highlight the relationship between the epigenome and transcriptome.
Authors: Schiffino FL, Siemian JN, Petrella M, Laing BT, Sarsfield S, Borja CB, Gajendiran A, Zuccoli ML, Aponte Y
Journal: PLoS One. 2019 Jul 10;14(7):e0219522. doi: 10.1371/journal.pone.0219522. eCollection 2019.
Across species, motivated states such as food-seeking and consumption are essential for survival. The lateral hypothalamus (LH) is known to play a fundamental role in regulating feeding and reward-related behaviors. However, the contributions of neuronal subpopulations in the LH have not been thoroughly identified. Here we examine how lateral hypothalamic leptin receptor-expressing (LHLEPR) neurons, a subset of GABAergic cells, regulate motivation in mice. We find that LHLEPR neuronal activation significantly increases progressive ratio (PR) performance, while inhibition decreases responding. Moreover, we mapped LHLEPR axonal projections and demonstrated that they target the ventral tegmental area (VTA), form functional inhibitory synapses with non-dopaminergic VTA neurons, and their activation promotes motivation for food. Finally, we find that LHLEPR neurons also regulate motivation to obtain water, suggesting that they may play a generalized role in motivation. Together, these results identify LHLEPR neurons as modulators within a hypothalamic-ventral tegmental circuit that gates motivation.