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Abstract Glycosylphosphatidylinositol-anchored proteins were reported to be involved in many developmental progresses in Arabidopsis. Here I report that, a group of homologous glycosylphosphatidylinositol-anchored proteins from SKU5-Similar family regulate seedling root development of Arabidopsis through controlling cell polar expansion and cell wall synthesis. Due to the irregular expansion of root cells and the defective synthesis of cell walls, their knockout mutants generated shorter roots with irregularly shaped root cells, and thicker cell walls. Highlights • SKS1, SKS2, SKS3 and SKU5 from SKS family are GPI-anchored proteins. SKS1 , SKS3 and SKU 5 could be transcribed during root development of Arabidopsis, and they are essential for cell polar expansion and cell wall synthesis. [ABSTRACT FROM AUTHOR]

The toxin-antitoxin system is ubiquitously existed in bacteria and archaea, performing a wide variety of functions modulating cell fitness in response to environmental cues. In this report, we solved the crystal structure of the toxin-antitoxin HigBA complex from E. coli K-12 to 2.7 Å resolution. The crystal structure of the HigBA complex displays a hetero-tetramer (HigBA) 2 form comprised by two HigB and two HigA subunits. Each toxin HigB resumes a microbial RNase T1 fold, characteristic of a three antiparallel β-sheet core shielded by a few α-helices at either side. Each antitoxin HigA composed of all α-helices resembles a “C”-shaped clamp nicely encompassing a HigB in the (HigBA) 2 complex. Two HigA monomers dimerize at their N-terminal domain. We showed that HigA helix α1 was essential for HigA dimerization and the hetero-tetramer (HigBA) 2 formation, but not for a hetero-dimeric HigBA formation. HigA dimerization mediated by helix α1 was dispensable for DNA-binding, as a heterodimeric HigBA complex still bound to the higBA operator in vitro . The HigA C-terminal domain with a helix-turn-helix fold was essential for DNA binding. We also defined two palindromes in higBA operator specifically recognized by HigA and HigBA in vitro . [ABSTRACT FROM AUTHOR]

Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae , which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance. [ABSTRACT FROM AUTHOR]

Abstract: Synaptotagmin I (Syt I) is a Ca2+ sensor for triggering fast synchronized release of neurotransmitters. However, controversy remains whether Syt I is also obligatory for the exocytosis and endocytosis of larger dense core vesicles (LDCVs) in endocrine cells. In this study, we used a short hairpin RNA (shRNA) to silence the expression of Syt I and investigated the roles of Syt I on exocytosis and endocytosis in INS-1 cells. Our results demonstrated that expression of Syt I is remarkably reduced by the Syt I gene targeting shRNA. Using high-time resolution capacitance measurement, we found that the silence of Syt I decreased the calcium sensitivity of fusion of insulin granules and therefore reduced the exocytotic burst triggered by step-like [Ca2+] i elevation. In addition, the occurrence frequency and amplitude of fast endocytosis were remarkably reduced in the silenced cells. We conclude that Syt I not only participates in the Ca2+-sensing of LDCV fusion with plasmalemma, but also plays a crucial role in fast endocytosis in INS-1 cells. [Copyright &y& Elsevier]

HigA functions as the antitoxin in HigB-HigA toxin-antitoxin system. It neutralizes HigB-mediated toxicity by forming a stable toxin-antitoxin complex. Here the crystal structure of isolated HigA from Escherichia coli str. K- 12 has been determined to 2.0 Å resolution. The structural differences between HigA and HigA in HigBA complex imply that HigA undergoes drastic conformational changes upon the binding of HigB. The conformational changes are achieved by rigid motions of N-terminal and C-terminal domains of HigA around its central linker domain, which is different from other known forms of regulation patterns in other organisms. As a transcriptional regulator, HigA bind to its operator DNA through the C-terminal HTH motif, in which key residues were identified in this study. • HigA forms a homodimer in solution by hydrophobic interactions of its N-ter domain. • HigA underwent drastic conformational changes upon binding HigB. • The conformational changes are rigid motions of domains around the linker. • The conformational changes facilitated the operator binding ability of HigBA complex [ABSTRACT FROM AUTHOR]

27-hydroxycholesterol (27-HC), the most abundant metabolite of cholesterol, is a risk factor for breast cancer. It can increase the proliferation of breast cancer cells and promote the metastasis of breast tumours in mouse models. Myc is a critical oncoprotein overexpressed in breast cancer. However, whether 27-HC affects Myc expression has not been reported. In the current study, we aimed to investigate the effects of 27-HC on Myc and the underlying mechanisms in MCF-7 breast cancer cells. Our data demonstrated that 27-HC activated Myc via increasing its protein stability. Three key negative modulators of Myc protein stability, PP2A, SCP1 and FBW7, were suppressed by 27-HC at the transcriptional level. We performed a data-mining analysis of the chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) data in the ChIPBase, and discovered that a number of putative transcription factors (TFs), including Myc itself, were involved in the transcriptional regulation of PP2A , SCP1 and FBW7 . Our results provide a novel mechanistic insight into the activation of Myc by 27-HC via transcriptional repression of PP2A , SCP1 and FBW7 to increase Myc protein stability in breast cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: Amiloride, a potassium sparing diuretic, is well known to interact with many ion transport systems and modulate the activity of several membrane receptors. However, relatively little information is available as to how amiloride affects membrane receptors of neurons in the brain areas. In the present study, we investigated the effects of amiloride on glycine-induced currents (I Gly) in cultured neurons of rat inferior colliculus with whole-cell patch-clamp recordings. Amiloride itself did not activate any current across the neuronal membrane but it reversibly inhibited the amplitude of the I Gly in a reversible and concentration-dependent manner, with an IC50 of 487.4±25.3μM (n =5). Amiloride shifted the concentration–response relationship to the right without changing Hill coefficient and without changing the maximum response of the I Gly. The pre-perfusion of amiloride produced an inhibitory effect on the I Gly. In addition, amiloride was shown with a voltage ramp protocol to significantly reduce the conductance induced by glycine but not to change the reversal potential of the I Gly. These results demonstrate that amiloride competitively inhibits the I Gly in rat inferior colliculus neurons by decreasing the affinity of glycine to its receptor. Our finding suggests that attention should be paid to the possible side effects of amiloride used as a drug on brain functions in the case of a defective blood–brain barrier and in the case of direct application of this drug into the cerebrospinal fluid for treatment of brain tumors. [Copyright &y& Elsevier]

HD-domain is a conserved domain, with the signature of histidine and aspartic (HD) residues doublets. HD-domain proteins may possess nucleotidase and phosphodiesterase activities, and they play important roles in signaling and nucleotide metabolism. In yeast, HD-domain proteins with nucleotidase activity remained unexplored. Here, we biochemically and structurally characterized two HD domain proteins YGK1 (YGL101W) and YB92 (YBR242W) from Saccharomyces cerevisiae as nucleoside 5ʹ-monophosphatases, with substrate preference for deoxyribonucleoside 5ʹ-monophosphatase over ribonucleoside 5ʹ-monophosphatase. By determining the crystal structure of YGK1, we unveiled that YGK1 structure resembled as the crystal structure of YfbR from E. coli . Size-exclusion chromatography and crosslinking studies suggested that YGK1 and YB92 existed in the form of a dimer, respectively, which were consistent with structural observation of YGK1. Site-directed mutagenesis demonstrated that more extensive conserved residues near the divalent metal coordinating active site were essential for YGK1 activity than previous suggested. The metal coordinating His89 and Asp90, and the neighboring conserved Glu93, Glu114 and Glu145 were individually critical for catalysis. In addition, alignments suggested that three flexible loops with hydrophobic residues might be implicated in substrate selectivity to nucleoside moiety. Together, our comparative structural and mutational studies suggested that YGK1 and YB92 functioned as 5ʹ-nucleotidases in S. cerevisiae . [ABSTRACT FROM AUTHOR]

In hearing loss induced by aminoglycoside antibiotics, the outer hair cells (OHCs) in the basal turn are always more susceptible than OHCs in the apical turn, while the underlying mechanisms remain unknown. In this study, we reported that NAPDH oxidase 2 (NOX2) played an important role in the OHCs damage preferentially in the basal turn. Normally, NOX2 was evenly expressed in OHCs among different turns, at a relatively low level. However, after neomycin treatment, NOX2 was dominantly induced in OHCs in the basal turn. In vivo and in vitro studies demonstrated that inhibition of NOX2 significantly alleviated neomycin-induced OHCs damages, as seen from both the cleaved caspase-3 and TUNEL staining. Moreover, gp91 ds-tat delivery and DHE staining results showed that NOX2-derived ROS was responsible for neomycin ototoxicity. Taken together, our study shows that regional up-expression of NOX2 and subsequent increase of ROS in OHCs of the basal turn is an important factor contributing to the vulnerability of OHCs there, which should shed light on the prevention of hearing loss induced by aminoglycoside antibiotics. [ABSTRACT FROM AUTHOR]