Top 10 Biorxiv Papers Today in Biochemistry


2.053 Mikeys
#1. A cooperative network of molecular "hot spots" highlights the complexity of LH3 collagen glycosyltransferase activities
Antonella Chiapparino, Francesca De Giorgi, Luigi Scietti, Silvia Faravelli, Tony Roscioli, Federico Forneris
Hydroxylysine glycosylations are collagen-specific post-translational modifications essential for maturation and homeostasis of fibrillar as well as non-fibrillar collagen molecules. Lysyl hydroxylase 3 (LH3) is the only human enzyme capable of performing two chemically-distinct collagen glycosyltransferase reactions using the same catalytic site: inverting beta-1,O-galactosylation of hydroxylysines and retaining alpha-1,2-glycosylation of galactosyl hydroxylysines. Here, we used structure-based mutagenesis to show that both glycosyltransferase activities are strongly dependent on a broad cooperative network of amino acid side chains which includes the first-shell environment of the glycosyltransferase catalytic site and shares features with both retaining and inverting enzymes. We identified critical "hot spots" leading to selective loss of inverting activity without affecting the retaining reaction. Finally, we present molecular structures of LH3 in complex with UDP-sugar analogs which provide the first structural templates for...
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biorxivpreprint: A cooperative network of molecular "hot spots" highlights the complexity of LH3 collagen glycosyltransferase activities https://t.co/JCbO4SY38z #bioRxiv
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Authors: 6
Total Words: 0
Unqiue Words: 0

2.0 Mikeys
#2. ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer
Xiaoshan Shi, Adam L Yokom, Chunxin Wang, Lindsey N Young, Richard J Youle, James H Hurley
The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain electron microscopy (EM) and multiangle light scattering showed that FIP200 is a dimer whilst a single molecule each of the other subunits is present. The FIP200 NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13-ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structure similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy initiating TBK1 kinase and the ULK1 kinase complex.
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biorxivpreprint: ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer https://t.co/a6g6q4D3I5 #bioRxiv
cryoEM_Papers: ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer https://t.co/ij92WqDbP7
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Sample Sizes : [3, 2]
Authors: 6
Total Words: 12758
Unqiue Words: 3389

1.998 Mikeys
#3. Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina)
RAFAEL IGNACIO MONSALVE, RUTH GUTIERREZ, ILKA HOOF, MANUEL LOMBARDERO
The aim of this study was to purify potential allergenic components of Vespa velutina venom, the yellow legged Asian Hornet, and perform a preliminary characterization of the purified proteins. Starting from the whole venom of V.velutina , several chromatographic steps allowed to purify the phospholipase (named Vesp v 1), as well as the antigen 5 (Vesp v 5, the only allergenic component described as such so far). The two hyaluronidase isoforms found (Vesp v 2A and Vesp v 2B) cannot be separated from each other, but they are partially purified and characterized. Purity of the isolated proteins in shown by SDSPAGE, as well as by the results of the N-terminal sequencing. This characterization and nLC-MS/MS data provide most of the sequence for Vesp v 1 and Vesp v 5 (72 and 84% coverage, respectively), confirming that the whole sequences of the isolated natural components match with the data available in public transcriptomic databases. It is of particular interest that Vesp v 1 is a glycosylated phospholipase, a fact that had only...
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biorxivpreprint: Purification and molecular characterization of phospholipase, antigen 5 and hyaluronidases from the venom of the Asian hornet (Vespa velutina) https://t.co/Ehu62gdh9a #bioRxiv
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Authors: 4
Total Words: 6869
Unqiue Words: 1999

1.997 Mikeys
#4. PHGDH supports liver ceramide synthesis and sustains lipid homeostasis
Yun Pyo Kang, Aimee Falzone, Min Liu, James J Saller, Florian A Karreth, Gina M DeNicola
D-3-phosphoglycerate dehydrogenase (PHGDH), which encodes the first enzyme in serine biosynthesis, is overexpressed in human cancers and has been proposed as a drug target. However, whether PHGDH is critical for the proliferation or homeostasis of tissues following the postnatal period is unknown. To study PHGDH inhibition in adult animals, we developed a knock-in mouse model harboring a PHGDH shRNA under the control of a doxycycline-inducible promoter. With this model, PHGDH depletion can be globally induced in adult animals, while sparing the brain due to poor doxycycline delivery. We found that PHGDH depletion is well tolerated and no overt phenotypes were observed in multiple highly proliferative cell compartments. Further, despite detectable knockdown, liver and pancreatic function were normal. Interestingly, diminished PHGDH expression in the liver reduced serine and ceramide levels without increasing the levels of deoxysphingolipids. Further, liver triacylglycerol profiles were altered, with an accumulation of longer chain,...
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RutterLab: RT @biorxivpreprint: PHGDH supports liver ceramide synthesis and sustains lipid homeostasis https://t.co/ZtJZYbvsAO #bioRxiv
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Authors: 6
Total Words: 0
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1.997 Mikeys
#5. The effect of proximity on the function and energy transfer capability of fluorescent protein pairs
Jacob Pope, Rachel Johnson, William David Jamieson, Harley Worthy, Senthil Kailasam, Husam Auhim, Daniel Watkins, Pierre Rizkallah, Oliver Castell, Dafydd Jones
Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Forster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity...
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biorxivpreprint: The effect of proximity on the function and energy transfer capability of fluorescent protein pairs https://t.co/CkhECl0h4Z #bioRxiv
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Authors: 10
Total Words: 0
Unqiue Words: 0

1.997 Mikeys
#6. Mechanism of the secretion of the lanthipeptide nisin
Marcel Lagedroste, Jens Reiners, Sander HJ Smits, Lutz Schmitt
Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied example is nisin, which is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT which secrets nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the maturation machinery composed of the dehydratase NisB and the cyclase NisC. The data clearly demonstrated that the function of NisC, but the mere presence of NisB modulated the apparent secretion rates. Additional in vitro studies of detergent-solubilized NisT revealed how the activity of this ABC transporter is again influenced by the enzymes of the maturation machinery, but not the substrate.
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biorxivpreprint: Mechanism of the secretion of the lanthipeptide nisin https://t.co/iUZSp2hxZt #bioRxiv
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Authors: 4
Total Words: 15809
Unqiue Words: 4249

1.997 Mikeys
#7. Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA
Linjia Su, Nadezda Bryan, Sabrina Battista, Juliano Freitas, Alyssa Garabedian, Federica DAlessio, Miriam Romano, Fabiana Falanga, Alfredo Fusco, Lidia Kos, Jeremy Chambers, Francisco Fernandez-Lima, Prem P. Chapagain, Stefan Vasile, Layton Smith, Fenfei Leng
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein which plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen high throughput screening (HTS) assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we discovered that suramin, a negatively charged antiparasitic drug potently inhibits the HMGA2-DNA interaction. Our results also show that the inhibition is through suramin binding to the AT-hooks of HMGA2, therefore blocking its DNA binding capacity. Furthermore, we demonstrate that suramin can induce brain tumor stem cells differentiation into cells with neurite-like structures, a process triggered by disrupting HMGA2-DNA interactions. Since suramin has strong antitumor and anti-metastasis activities, our...
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Authors: 16
Total Words: 10489
Unqiue Words: 3466

1.997 Mikeys
#8. Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: M319C, T431I, and K337D
Peishan Huang, Stephaine C Contreras, Eliana Bloomfield, Kristine Schmitz, Augustine Arredondo, Justin B Siegel
The use of computational tools has become an increasingly popular tool for engineering protein function. While there are numerous examples of computational tools enabling the design of novel protein functions, there remains room for improvement in both prediction accuracy and success. To improve algorithms for functional and stability predictions, we have initiated the development of a data set designed to be used for training new computational algorithms for enzyme design. To date our dataset is composed of over 129 mutants with associated expression levels, kinetic data, and thermal stability for the enzyme β- glucosidase B (BglB) from Paenibacillus polymyxa. In this study, we introduced three new variants (M319C, T431I, and K337D) to our existing dataset with the goal of cultivating a larger dataset to train new design algorithms and more broadly explore structure-function relationships in BglB.
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Authors: 6
Total Words: 0
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1.997 Mikeys
#9. Secondary motifs enable concentration-dependent regulation by Rbfox family proteins
Bridget E Begg, Marvin Jens, Peter Y Wang, Christopher B Burge
The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity, but just half of Rbfox CLIP peaks contain a GCAUG motif. We incubated recombinant RBFOX2 with over 60,000 transcriptomic sequences to reveal significant binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX concentrations. We find that many of these secondary motifs bind Rbfox robustly in vivo and that several together can exert regulation comparable to a GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.
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Sample Sizes : [388, 561]
Authors: 4
Total Words: 15696
Unqiue Words: 3831

1.997 Mikeys
#10. The flexible N-terminus of BchL protects its [4Fe-4S] cluster in oxygenic environments and autoinhibits activity
Elliot I Corless, Syed Muhammad Saad Imran, Maxwell B Watkins, Sofia Origanti, John P Bacik, Robert Kitelinger, Mark S Soffe, Karamatullah Danyal, Lance C Seefeldt, Brian Bennett, Nozomi Ando, EDwin Antony
The dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosyn-thesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here pre-sent an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid sub-stitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stabil-ity. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp (DFD patch), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and...
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Authors: 12
Total Words: 0
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