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At least 235 records · Page 13Linked to original sources

bioRxiv2017

MmpL3 is the flippase for mycolic acids in mycobacteria

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which in part render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. While the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. MmpL3, an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several anti-mycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria, and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism-of-action of BM212 as a potent MmpL3 inhibitor, and employ it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future anti-tuberculosis drugs.

biochemistry↗

bioRxiv2017

Emergence of localized patterns in globally coupled networks of relaxation oscillators with heterogeneous connectivity

Relaxation oscillators may exhibit small amplitude oscillations (SAOs) in addition to the typical large amplitude oscillations (LAOs) as well as abrupt transitions between them (canard phenomenon). Localized cluster patterns in networks of relaxation oscillators consist of one cluster oscillating in the LAO regime or exhibiting mixed-mode oscillations (LAOs interspersed with SAOs), while the other oscillates in the SAO regime. We investigate the mechanisms underlying the generation of localized patterns in globally coupled networks of piecewise-linear (PWL) relaxation oscillators where global feedback acting on the rate of change of the activator (fast variable) involves the inhibitor (slow variable). We also investigate of these patterns are affected by the presence of a diffusive type of coupling whose synchronizing effects compete with the symmetry breaking global feedback effects.

biochemistry↗

bioRxiv2017

Internal modifications in the CENP-A nucleosome modulate centromeric dynamics

Post-translational modifications (PTMs) of core histones have studied for over 2 decades, and are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core, and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using an acetyl or unacetylatable mimic (CENP-A K124Q and K124A respectively) reveal alterations in CENP-C levels, and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing, with the permanently acetylated form replicating centromeres early, and the unacetylable form replicating centromeres late. Purification of native CENP-A proteins followed by mass spectrometry analysis reveal that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early and mid-S phase. Finally, we provide evidence that the HAT p300 is involved in this cycle. Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome can influence the binding of key kinetochore proteins, the integrity of mitosis and centromeric replication. These data support the emerging paradigm that core modifications in histone variant nucleosomes transduce defined changes to key biological processes.

biochemistry↗

bioRxiv2017

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands

AbstractRecent data have expanded our understanding of Notch signaling by identifying a C2 domain at the N-terminus of Notch ligands which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and DLL4 and human Notch-2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signaling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations which affect these loops and are associated with extrahepatic biliary atresia lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signaling in different physiological contexts.

biochemistry↗

bioRxiv2017

DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute

We show here that, unlike most other prokaryotic Argonaute (Ago) proteins, which are DNA-guided endonucleases, the Natronobacterium gregoryi-derived Ago (NgAgo) can function as a DNA-guided endoribonuclease, cleaving RNA, rather than DNA, in a targeted manner. The NgAgo protein, in complex with 5-hydroxylated or 5-phosphrylated oligodeoxyribonucleotides (ODNs) of variable lengths, split RNA targets into two or more fragments in vitro, suggesting its physiological role in bacteria and demonstrating a potential for degrading RNA molecules such as mRNA or lncRNA in eukaryotic cells in a targeted manner.

biochemistry↗

bioRxiv2017

Proteogenomics of adenosine-to-inosine RNA editing in fruit fly

Adenosine-to-inosine RNA editing is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. We present a proteomic study on this phenomenon for Drosophila melanogaster. Three proteome data sets were used in the study: two taken from public repository and the third one obtained here. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies and applied for identifying the edited proteins. The total number of 68 edited peptides belonging to 59 proteins was identified in all data sets. Eight of them being shared between the whole insect, head and brain proteomes. Seven edited sites belonging to synaptic vesicle and membrane trafficking proteins were selected for validation by orthogonal analysis by Multiple Reaction Monitoring. Five editing events in cpx, Syx1A, Cadps, CG4587 and EndoA were validated in fruit fly brain tissue at the proteome level using isotopically labeled standards. Ratios of unedited-to-edited proteoforms varied from 35:1 (Syx1A) to 1:2 (EndoA). Lys-137 to Glu editing of endophilin A may have functional consequences for its interaction to membrane. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.

biochemistry↗

bioRxiv2017

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A

Vascular Endothelial Growth Factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single particle electron microscopy, small angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here we describe the structure of the full-length VEGFR-1 extracellular domain (ECD) in complex with VEGF-A at 4 [A] resolution. We combined X-ray crystallography, single particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in Ig-domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

biochemistry↗

bioRxiv2017

Cell proliferation depends on the direct binding between PKM2 and AKAP-Lbc

The M2 form of the glycolytic enzyme pyruvate kinase (PKM2) has generated much interest recently due to its important role in tumor metabolism. A yeast two-hybrid screen carried out by the Alliance for Cell Signaling suggests that PKM2 interacts with A-Kinase Anchoring Protein (AKAP)-Lbc.\n\nAKAP-Lbc (also known as AKAP13) is a scaffold protein that integrates signaling through multiple enzymes including protein kinases A and D and the small G protein Rho. AKAP-Lbc was originally identified in leukemic blast cells, and multiple reports implicate AKAP-Lbc in breast, prostate and thyroid cancers, however the role of AKAP-Lbc in cancer biology is not understood.\n\nCo-immunoprecipitation, pulldown and Bimolecular Fluorescence Complementation (BiFC) data indicate that PKM2 interacts with AKAP-Lbc. Mapping experiments indicate that PKM2 directly interacts with amino acid residues 1923-2817 of AKAP-Lbc. By disrupting the interaction between the two proteins with the expression of the AKAP-Lbc fragments, our data suggest that the binding between PKM2 and PKA plays a critical role in cell proliferation. The work indicates that the binding between AKAP-Lbc and PKM2 may be an important target to treat some cancers by reducing the cell proliferation.

biochemistry↗

bioRxiv2017

Remodeling Pathway Control of Oxidative Phosphorylation by Temperature in the Heart

The capacity of mitochondrial oxidative phosphorylation (OXPHOS) and fuel substrate supply are key determinants of cardiac muscle performance. Although temperature exerts a strong effect on energy metabolism, until recently numerous respiratory studies of mammalian mitochondria have been carried out below physiological temperature, with substrates supporting submaximal respiratory capacity. We measured mitochondrial respiration as a function of temperature in permeabilized fibers from the left ventricle of the mouse heart. At 37 {degrees}C, OXPHOS capacity with electron entry through either Complex I or Complex II into the Q-junction was about half of respiratory capacity with the corresponding physiological substrate combination reconstituting tricarboxylic acid cycle function with convergent electron flow through the NADH&succinate (NS) pathway. When separating the component core mitochondrial pathways, the relative contribution of the NADH pathway increased with a decrease of temperature from 37 to 25 {o}C. The additive effect of convergent electron flow has profound consequences for optimization of mitochondrial respiratory control. The apparent excess capacity of cytochrome c oxidase (CIV) was 0.7 above convergent NS-pathway capacity, but would be overestimated nearly 2-fold with respect to respiration restricted by provision of NADH-linked substrates only. The apparent excess capacity of CIV increased sharply at 4 {degrees}C, caused by a strong temperature dependence of and OXPHOS limitation by NADH-linked dehydrogenases. This mechanism of mitochondrial respiratory control in the hypothermic mammalian heart is comparable to the pattern in ectotherm species, pointing towards NADH-linked mt-matrix dehydrogenases and the phosphorylation system rather than electron transfer complexes as the primary drivers of thermal sensitivity at low temperature and likely modulators of temperature adaptation and acclimatization. Delineating the link between stress and remodeling of OXPHOS is critically important for improving our understanding of metabolic perturbations in disease evolution and cardiac protection. Temperature is not a trivial experimental parameter to consider when addressing these questions.

biochemistry↗

bioRxiv2017

Cannabinoid glycosides: In vitro production of a new class of cannabinoids with improved physicochemical properties

The cannabinoid signaling system has recently garnered attention as a therapeutic target for numerous indications, and cannabinoids are now being pursued as new treatment options in diverse medical fields such as neurology, gastroenterology, pain management, and oncology. Cannabinoids are extremely hydrophobic and relatively unstable compounds, and as a result, formulation and delivery options are severely limited. Enzymatic glycosylation is a strategy to alter the physicochemical properties of small molecules, often improving their stability and aqueous solubility, as well as enabling site-specific drug targeting strategies. To determine if cannabinoids are a candidate for glycosylation, a library of glucosyltransferase (UGT) enzymes was screened for glycosylation activity towards various cannabinoids. The UGT76G1 enzyme from Stevia rebaudiana has been identified as having glucosyltransferase activity towards a broad range of cannabinoids. Compounds that were successfully glycosylated by UGT76G1 include the phytocannabinoids cannabidiol (CBD), {Delta}9-tetrahydrocannabinol ({Delta}9-THC), cannabidivarin (CBDV), and cannabinol (CBN), and the human endocannabinoids anandamide (AEA), 2-arachidonoyl-glycerol (2AG), 1-arachidonoyl-glycerol (1AG), and synaptamide (DHEA). Interestingly, UGT76G1 is able to transfer primary, secondary, and tertiary glycosylations at each acceptor of most of the cannabinoids tested. Additionally, Os03g0702000p, a glycosyltransferase from Oryza sativa, was able to transfer secondary glucose residues onto cannabinoid monoglycosides previously established by UGT76G1. This new class of cannabinoid-glycosides has been termed cannabosides. The compounds have greatly improved solubility in aqueous solutions. This increased aqueous solubility may enable new oral pharmaceutical delivery options for cannabinoids, as well as targeted delivery and release of cannabinoids within the intestines through glycoside prodrug metabolism.

biochemistry↗

bioRxiv2017

Releasing potential power of terpene synthases by a robust precursor supply platform

Approximately 76,000 discovered makes terpenoids the largest family of natural products in nature with widespread applications. The wide-spectrum of structural diversity of the terpenoids were largely due to the variable skeletons generated by terpene synthases. The number of terpene skeletons found in nature, however, were so much more than those conceivably generated from known terpene synthases and the limited characterized terpene synthases also make no chance for some useful terpenoids overproduction in microbe. Here, we first demonstrated that the promiscuous synthases in vivo can produce more variable terpenoid products by converting precursors of different lengths (C10, C15, C20, C25). This discovery was prompted by the development of an efficient in vivo platform by combining the two promiscuous terpene synthases and three prenyltransferases to generate 50 terpenoids, at least 3 ring systems of which were completely new. Furthermore, protein engineering was further integrated to enhance product diversity. Clearly, the work is expected to dramatically reshape the terpenoid research by widening the flexibility of the terpene synthases for the fresh discovery or creation of the new terpenoid compounds by skeleton reframing.

biochemistry↗

bioRxiv2017

The Dense-Core Vesicle Maturation Protein CCCP-1 Binds both RAB-2 and Membranes through a Conserved C-terminal Coiled-coil Domain

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.\n\nSynopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

biochemistry↗

bioRxiv2017

Structure of a AAA+ unfoldase in the process of unfolding substrate

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 [A] stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

biochemistry↗

bioRxiv2017

Versatile Multicolor Nanodiamond Probes for Intracellular Imaging and Targeted Labeling

Diamond nanoparticles that host bright luminescent centers are attracting attention for applications in bio-labeling and bio-sensing. Beyond their unsurpassed photostability, diamond can host multiple color centers, from the blue to the near infra-red spectral range. While nanodiamonds hosting nitrogen vacancy defects have been widely employed as bio-imaging probes, production and fabrication of nanodiamonds with other color centers is a challenge. In this work, a large scale production of fluorescent nanodiamonds (FNDs) containing a near infrared (NIR) color center - namely the silicon vacancy (SiV) defect, is reported. More importantly, a concept of application of different color centers for multi-color bio-imaging to investigate intercellular processes is demonstrated. Furthermore, two types of FNDs within cells can be easily resolved by their specific spectral properties, where data shows that SiV FNDs initially dispersed throughout the cell interior while NV FNDs localized in a close proximity to nucleus. The reported results are the first demonstration of multi-color labeling with FNDs that can pave the way for the wide-ranging use of FNDs in applications, including bio-sensing, bio-imaging and drug delivery applications.

biochemistry↗

bioRxiv2017

Translation efficiency is maintained at elevated temperature in E. coli

Cellular protein levels are dictated by the balance between gene transcription, mRNA translation and protein degradation, among other factors. Cells must manage their proteomes during stress; one way in which they may do so, in principle, is by differential translation. We used ribosome profiling to directly monitor translation in E. coli at 30 {degrees}C and investigate how this changes after 10-20 minutes of heat shock at 42 {degrees}C. Translation is controlled by the interplay of several RNA hybridization processes, which are expected to be temperature sensitive. However, translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat shock response transcriptional program at 30 {degrees}C. Several gene-specific parameters correlated with translation efficiency, including predicted mRNA structure and whether a gene is cotranslationally translocated into the inner membrane. Genome-wide predictions of the temperature dependence of mRNA structure suggest that relatively few genes show a melting transition between 30 {degrees}C and 42 {degrees}C, consistent with our observations. A linear model with five parameters can predict 33% of the variation in translation efficiency between genes, which may be useful in interpreting transcriptome data.

biochemistry↗

bioRxiv2017

Atomic resolution structure of serine protease proteinase K at ambient temperature

Atomic resolution structures (beyond 1.20 [A]) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 [A] resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

biochemistry↗

bioRxiv2017

PGC-1α coordinates with Bcl-2 to control cell cycle in U251 cells through reducing ROS

B-cell lymphoma 2 (Bcl-2) has a dual function, acting both as an oncogene and an anti-tumor gene. It is well known that Bcl-2 exerts its tumor promoting function through the mitochondrial pathway. However, the mechanism by which Bcl-2 suppresses tumor formation is not well understood. We have previously shown that Bcl-2 inhibits cell cycle progression from the G0/G1 to the S phase after serum starvation, and that quiescent Bcl-2 expressing cells maintained a significant lower level of mitochondrial reactive oxygen species (ROS) than the control cells. Based on the fact that ROS mediate cell cycle progression, and are controlled by peroxisome proliferator-activated receptor-{gamma} co-activator 1 (PGC-1), a key molecule induced by prolonged starvation and involved in mitochondrial metabolism, we hypothesized that PGC-1 might be related with the cell cycle function of Bcl-2. Here, we showed that PGC-1 was upregulated upon Bcl-2 overexpression and downregulated following Bcl-2 knockdown during serum starvation. Knockdown of PGC-1 activated Bcl-2 expression. Taken together, our results suggest that after serum depletion, PGC-la might coordinate with Bcl-2 to reduce ROS, which in turn delay cell cycle progression.\n\nSummary statementPGC-1 coordinate with Bcl-2 delay cell cycle progression to reduce ROS after serum depletion in human glioma U251 cells.

biochemistry↗

bioRxiv2017

Identifying a TFIID interactome via integrated biochemical and high-throughput proteomic studies

The core promoter recognition TFIID complex acts as a central regulator for eukaryotic gene expression. To direct transcription initiation, TFIID binds the core promoter DNA and aids recruitment of the transcription machinery (e.g., RNA polymerase II) to the transcription start site. Many transcription factors target TFIID to control vital cellular processes. Current studies on finding TFIID interactors have predominantly focused on transcription factors. Yet, a comprehensive interactome of mammalian TFIID has not been established. Therefore, this study sought to reveal potential TFIID-nucleated networks by identifying likely co-regulatory factors that bind TFIID. By using intact native human TFIID complexes, we have exploited three independent approaches including a high-throughput Next Generation DNA sequencing coupled with proteomic analysis. Among these methods, we found some overlapping and new candidates in which we further assessed three putative interactors (i.e., Sox2, H2A and EMSY) by co-immunoprecipitation assays. Notably, in addition to known TFIID interactors, we identified a number of novel factors that participate either in co-regulatory pathways or non-transcription related functions of TFIID. Overal, these results indicate that, in addition to transcription initiation, mammalian TFIID may be involved in broader regulatory pathways than previous studies suggested.

biochemistry↗