Outer-membrane translocation of bulky small molecules by passive diffusion
Bert van den Berg, Satya Prathyusha Bhamidimarri, Jigneshkumar Dahyabhai Prajapati, Ulrich Kleinekathöfer, and Mathias Winterhalter
The outer membrane (OM) of gram-negative bacteria forms a protective layer on the outside of the cell that prevents unrestricted access of harmful compounds. For the acquisition of ions and nutrients, the OM contains two types of transport proteins: passive diffusion channels and active transporters. Due to the limited diameters of passive diffusion channels, bulky molecules such as iron–siderophores and complex oligosaccharides are assumed to be taken up exclusively by active transporters. Here (pp. E2991–E2999) we assert that this assumption is incorrect. Using a combination of biophysical and computational approaches, we show that the OM protein CymA (cyclodextrin metabolism A) from Klebsiella oxytoca represents a previously unidentified paradigm in OM transport by mediating the passive diffusion of cyclic oligosaccharides (cyclodextrins) with diameters of ∼15 Å.
Human ortholog of Drosophila Melted impedes SMAD2 release from TGF-β receptor I to inhibit TGF-β signaling
Premalatha Shathasivam, Alexandra Kollara, Maurice J. Ringuette, Carl Virtanen, Jeffrey L. Wrana, and Theodore J. Brown
Ventricular zone expressed pleckstrin homology domain-containing 1 (VEPH1) is among genes on chromosome 3q24-26, a region amplified in several cancers. Although little is known of mammalian VEPH1, its Drosophila ortholog, Melted, is involved in neural and eye development, metabolism, and size determination through effects on Forkhead box O, target of rapamycin, and Hippo signaling. We show that VEPH1 expression affects similar gene categories as Melted and potently inhibits transforming growth factor-β (TGF-β) signaling. VEPH1 interacts with TGF-β type I receptor (TβRI) and inhibits dissociation of activated Sma- and Mad-related protein 2 from TβRI, resulting in impaired TGF-β signaling. TGF-β acts initially as a tumor suppressor through its cytostatic activity, but subsequently promotes tumor progression. These findings (pp. E3000–E3009) suggest that VEPH1 could affect TGF-β activity during cancer development/progression.
Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity
Cynthia C. Jung, Denize Atan, David Ng, Lynda Ploder, Sarah E. Ross, Martin Klein, David G. Birch, Eduardo Diez, and Roderick R. McInnes
Knowledge of the molecules that guide retinal interneuron formation is incomplete. We showed that PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) is required for the development of rod bipolar cells and OFF-cone bipolar subtypes as well as amacrine cell identity. Although bipolar cells were specified in Prdm8-null mice, rod bipolar cell differentiation was impaired, leading to their death and near absence from adult retina. This defect disrupts postphotoreceptor signal transduction, as shown by nonprogressive b-wave deficits in electroretinograms. Our findings (pp. E3010–E3019) suggest PRDM8 as a candidate gene for human congenital stationary night blindness. They also establish PRDM8 as a component of the regulatory network governing bipolar cell development and amacrine cell diversity, aiding efforts to generate these essential interneurons in vitro.
hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function
Junqiang Ye, Nadine Beetz, Sean O’Keeffe, Juan Carlos Tapia, Lindsey Macpherson, Weisheng V. Chen, Rhonda Bassel-Duby, Eric N. Olson, and Tom Maniatis
We studied the physiological function of the heterogeneous nuclear ribonucleoprotein U (hnRNP U) by generating a conditional knockout mouse in which the Hnrnpu gene is deleted in the heart. We found that hnRNP U is required for normal pre-mRNA splicing and postnatal heart development and function. Mutant mice develop severe dilated cardiomyopathy and die 2 wk after birth. Phenotypic characterization of mutant hearts coupled with RNA-seq data analyses revealed that mutant hearts display multiple cardiac defects as a result of misregulated gene expression and abnormal pre-mRNA splicing. We also identified the sarcoplasmic reticulum membrane protein Junctin as a splicing target of hnRNP U and provide an interesting example of alternative splicing in controlling the modification and function of proteins (pp. E3020–E3029).
Angiopoietin-like 4 is a potent angiogenic factor and a novel therapeutic target for patients with proliferative diabetic retinopathy
Savalan Babapoor-Farrokhran, Kathleen Jee, Brooks Puchner, Syed Junaid Hassan, Xiaoban Xin, Murilo Rodrigues, Fabiana Kashiwabuchi, Tao Ma, Ke Hu, Monika Deshpande, Yassine Daoud, Sharon Solomon, Adam Wenick, Gerard A. Lutty, Gregg L. Semenza, Silvia Montaner, and Akrit Sodhi
In proliferative diabetic retinopathy (PDR), the most vision-threatening sequela of diabetic eye disease, retinal ischemia leads to increased expression of angiogenic factors that promote neovascularization. Although therapies targeting the potent angiogenic mediator vascular endothelial growth factor have been remarkably successful for the treatment of diabetic macular edema, this approach has not proven sufficient to prevent the development of retinal neovascularization, implicating additional angiogenic factor(s) in PDR pathogenesis. We demonstrate here (pp. E3030–E3039) that angiopoietin-like 4 is a potent angiogenic mediator with markedly increased expression in the eyes of PDR patients. Our studies identify a novel therapeutic target for the treatment of ocular neovascular disease and may have broad implications for the treatment of other diseases dependent on pathologic angiogenesis.
Regulation of age-related macular degeneration-like pathology by complement factor H
Christopher B. Toomey, Una Kelly, Daniel R. Saban, and Catherine Bowes Rickman
Age-related macular degeneration (AMD) affects approximately one-third of Americans over 70 and is characterized by lipoprotein-rich sub-retinal pigmented epithelium (sub-RPE) deposits. Substantial evidence has emerged that implicates complement factor H (CFH) in the pathogenesis of AMD. Here (pp. E3040–E3049), we conduct an in vivo analysis to elucidate the role of CFH in AMD pathology. We show that (i) CFH and lipoproteins compete for binding in the sub-RPE extracellular matrix such that decreasing CFH leads to lipoprotein accumulation and sub-RPE deposit formation; and (ii) derimental complement activation within sub-RPE deposits leads to RPE damage and vision loss. This new understanding of the complicated interactions of CFH in development of AMD-like pathology paves the way for identifying more targeted therapeutic strategies for AMD.
Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy
Christian L. Barrett, Christopher DeBoever, Kristen Jepsen, Cheryl C. Saenz, Dennis A. Carson, and Kelly A. Frazer
Identifying molecules that are specific to tumors for use in early detection, diagnosis, prognosis, and therapy is both a primary goal and a key discovery challenge across diverse areas of oncology. To discover ovarian tumor-specific molecules, we developed custom bioinformatics algorithms to analyze transcriptome sequence data of 296 ovarian cancer and 1,839 normal tissues and validated putative tumor-specific mRNA isoforms by RT–quantitative PCR. The results (pp. E3050–E3057) revealed multiple candidate diagnostic and therapeutic targets with unique sequences that were expressed in most of the cancers examined but not in normal tissues. The process we developed can be readily applied to identify diagnostic and therapeutic targets for any of the 30 or more tumor types for which large amounts of transcriptome data now exist.
Structure of a bacterial toxin-activating acyltransferase
Nicholas P. Greene, Allister Crow, Colin Hughes, and Vassilis Koronakis
Secreted pore-forming toxins of pathogenic bacteria such as Escherichia coli and Bordetella pertussis insert into cell membranes to subvert signaling and cause cell death, facilitating infection of human and animal hosts. These toxins require a unique activation step before secretion, the covalent linkage of lipid groups to specific lysines of the inactive protoxin, directed by a specialized toxin-activating acyl transferase (TAAT). Here (pp. E3058–E3066), we present the TAAT crystal structure, the soluble dimeric topology, and likely active site, revealing that despite no discernible sequence similarity, TAATs are a structurally and functionally distinct group of the Gcn5-like N-acetyl transferase (GNAT) superfamily of modifying enzymes. Our findings open the way to further understanding of the unique toxin activation, and the possibility of inhibiting toxin action.
BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met
Toshiyuki Mizui, Yasuyuki Ishikawa, Haruko Kumanogoh, Maria Lume, Tomoya Matsumoto, Tomoko Hara, Shigeto Yamawaki, Masami Takahashi, Sadao Shiosaka, Chiaki Itami, Koichi Uegaki, Mart Saarma, and Masami Kojima
Brain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits biological effects on synaptic plasticity. BDNF is initially synthesized as precursor proBDNF, and then the BDNF pro-peptide is simultaneously produced from the precursor protein. However, the physiological functions of the pro-peptide are largely unknown. Here (pp. E3067–E3074), we demonstrate that the BDNF pro-peptide is a facilitator of hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA-type receptors and the pan-neurotrophin receptor p75NTR. Second, a common BDNF polymorphism substitutes valine for methionine at amino acid position 66 (Val66Met) in the pro-peptide of BDNF and impairs memory function. Unexpectedly, the pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings provide insights into the physiological role of the BDNF pro-peptide in the brain.
Reduced endogenous Ca2+ buffering speeds active zone Ca2+ signaling
Igor Delvendahl, Lukasz Jablonski, Carolin Baade, Victor Matveev, Erwin Neher, and Stefan Hallermann
Calcium influx during action potentials triggers neurotransmitter release at presynaptic active zones. Calcium buffers limit the spread of calcium and restrict neurotransmitter release to the vicinity of calcium channels. To sustain synchronous release during repetitive activity, rapid removal of calcium from the active zone is essential, but the underlying mechanisms are unclear. Therefore, we focused on cerebellar mossy fiber synapses, which are among the fastest synapses in the mammalian brain and found very weak presynaptic calcium buffering. One might assume that strong calcium buffering has the potential to efficiently remove calcium from active zones. In contrast, our results (pp. E3075–E3084) show that weak calcium buffering speeds active zone calcium clearance. Thus, the strength of presynaptic buffering limits the rate of synaptic transmission.