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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 89,042 bioRxiv papers from 381,669 authors.

Most downloaded bioRxiv papers, all time

in category biochemistry

2,880 results found. For more information, click each entry to expand.

1801: Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity.
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Posted to bioRxiv 03 Oct 2018

Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity.
261 downloads biochemistry

Corey D. Seacrist, Raymond D. Blind

Inositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2 (PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (deltaIPMK) are presented. Although the KM for PIP2 is identical between deltaIPMK and wild type, deltaIPMK has a 1.8-fold increase in kcat for PIP2, indicating the native IPMK disordered domains decrease IPMK activity in vitro. The 2.5 angstrom crystal structure of deltaIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative ATP-clamp in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases the KM for ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

1802: Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases
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Posted to bioRxiv 08 May 2018

Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases
261 downloads biochemistry

Assou El-Battari, Sylvie Mathieu, Romain Sigaud, Maëlle Prorok-Hamon, L’Houcine Ouafik, Charlotte Jeanneau

The Golgi resident glycosyltransferases (GTs) are membrane-bound glycoproteins but are frequently found as soluble proteins in biological fluids where their function remains largely unknown. Previous studies have established that the release of these proteins involved Alzheimer disease-associated proteases such as β-secretases (BACE1 and BACE2) and the intramembrane-cleaving aspartyl proteases Presenilins 1 and 2. Recent studies have involved another intramembrane-cleaving enzyme, the signal peptide peptidese-like-3 (SPPL3). Except for the latter, the two former studies mostly addressed particular cases of GTs, namely ST6Gal-I (BACEs) or GnT-V (Presenilins). Therefore the question still remains as which of these secretases is truly responsible for the cleavage and secretion of GTs. We herein combined the 3 proteases in a single study with respect to their abilities to release 3 families of GTs encompassing three N-acetylglucosaminyltransferases, two fucosyltransferases and two sialyltransferases. Green fluorescent protein (gfp)-fused versions of these GTs were virally transduced in mouse embryonic fibroblasts devoid of BACEs, Presenilins or SPPL3. We found that neither BACE nor Presenilins are involved in the shedding of these glycosyltransferases, while SPPL3 was involved in the cleavage and release of some but not all GTs. Notably, the γ-secretase inhibitor DFK-167 was the only molecule capable of significantly decreasing glycosyltransferase secretion, suggesting the involvement of γ-secretase(s), yet different from Presenilins but comprising SPPL3 among other proteases still to be identified. Using confocal microscopy, we show that SPPL3 selectivity towards GTs relays not only on sequence specificity but also depends on how GTs distribute in the cell with respect SPPL3 during their cycling within and outside the Golgi.

1803: A conserved D/E-P motif in the nucleotide binding domain of plant ABCB/PGP-type ABC transporters defines their auxin transport capacity
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Posted to bioRxiv 09 May 2020

A conserved D/E-P motif in the nucleotide binding domain of plant ABCB/PGP-type ABC transporters defines their auxin transport capacity
261 downloads biochemistry

Pengchao Hao, Jian Xia, Jie Liu, Martin diDonato, Konrad Pakula, Aurélien Bailly, Michal Jasinski, Markus Geisler

Auxin transport activity of ABCB1 was suggested to be regulated by physical interaction with the FKBP42/Twisted Dwarf1 (TWD1), a bona fide peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity on TWD1 have failed so far. By using a structure-based approach we have identified a series of surface-exposed proline residues in the C- terminal nucleotide binding fold and linker of Arabidopsis ABCB1 that do not alter ABCB1 protein stability or location but its catalytic transport activity. P1.008 was uncovered as part of a conserved signature D/E-P motif that seems to be specific for Auxin-Transporting ABCBs, we now refer to as ATAs. Beside the proline, also mutation of the acidic moiety prior to the proline abolishes auxin transport activity by ABCB1. So far, all higher plant ABCBs for that auxin transport was safely proven carry this conserved motif underlining its diagnostic potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E- P1.008 motif is also important for ABCB1-TWD1 interaction and activation of ABCB1-mediated auxin transport by TWD1, supporting a scenario in that TWD1 acts as an activator of ABCB1 transport activity by means of its PPIase. In summary, our data imply a dual function for TWD1 acting as an ABCB co-chaperone required for ABCB biogenesis and as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds. ### Competing Interest Statement The authors have declared no competing interest.

1804: Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements
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Posted to bioRxiv 02 Nov 2019

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements
261 downloads biochemistry

Rory Henderson, Maolin Lu, Ye Zhou, Zekun Mu, Robert Parks, Qifeng Han, Allen L. Hsu, Elizabeth Carter, Scott C Blanchard, RJ Edwards, Kevin Wiehe, Kevin O. Saunders, Mario J Borgnia, Alberto Bartesaghi, Walther Mothes, Barton F Haynes, Priyamvada Acharya, S. Munir Alam

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, we introduced Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis performed on the soluble ectodomain BG505 SOSIP Env trimers, cell-surface expressed BG505 full-length trimers and single-molecule Forster Resonance Energy Transfer (smFRET) performed on the full-length virion-bound Env confirmed that these mutations prevented CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins revealed rearrangements in the gp120 topological layer contacts with gp41. Specifically, a conserved tryptophan at position 571 (W571) was displaced from its typical pocket at the interface of gp120 topological layers 1 and 2 by lysine 567, disrupting key gp120- gp41 contacts and rendering the Env insensitive to CD4 binding. Vector based analysis of closed Env SOSIP structures revealed the newly designed trimers exhibited a quaternary structure distinct from that typical of SOSIPs and residing near a cluster of Env trimers bound to vaccine-induced fusion peptide-directed antibodies (vFP Mabs). These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.

1805: Molecular Architecture of the Bardet-Biedl Syndrome Protein 2-7-9 Subcomplex
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Posted to bioRxiv 11 Jul 2019

Molecular Architecture of the Bardet-Biedl Syndrome Protein 2-7-9 Subcomplex
261 downloads biochemistry

W. Grant Ludlam, Takuma Aoba, Jorge Cuéllar, M. Teresa Bueno-Carrasco, Aman Makaju, James D. Moody, Sarah Franklin, José M. Valpuesta, Barry M. Willardson

Bardet-Biedl syndrome (BBS) is a genetic disease caused by mutations that disrupt the function of the BBSome, an eight-subunit complex that plays an important role in transport of proteins in primary cilia. To better understand the molecular basis of the disease, we analyzed the structure of a BBSome subcomplex consisting of three homologous BBS proteins (BBS2, BBS7, and BBS9) by an integrative structural modeling approach using electron microscopy and chemical crosslinking coupled with mass spectrometry. The resulting molecular model revealed an overall structure that resembles a flattened triangle. Within the structure, BBS2 and BBS7 form a tight dimer based on a coiled-coil interaction, and BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-linked mutation of BBS2 (R632P) is located in the α-helical domain at the interface between BBS2 and BBS9, and binding experiments showed that this mutation disrupted the interaction of BBS2 with BBS9. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing a molecular explanation for BBS in patients harboring this mutation.

1806: Dscam homophilic specificity is generated by high order cis-multimers coupled with trans self-binding of variable Ig1 in Chelicerata
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Posted to bioRxiv 16 Dec 2019

Dscam homophilic specificity is generated by high order cis-multimers coupled with trans self-binding of variable Ig1 in Chelicerata
260 downloads biochemistry

Fengyan Zhou, Guozheng Cao, Songjun Dai, Guo Li, Hao Li, Zhu Ding, Shouqing Hou, Bingbing Xu, Wendong You, Feng Shi, Xiaofeng Yang, Yongfeng Jin

By alternative splicing, Drosophila Down syndrome cell adhesion molecule (Dscam1) encodes tens of thousands of proteins required for establishing neural circuits, while Chelicerata encodes a family of ~ 100 shortened Dscam (sDscam) isoforms via alternative promoters. We report that Dscam isoforms interact promiscuously in cis to generate a vast repertoire of combinatorial homophilic recognition specificities in Chelicerata. Specifically, sDscams formed high order cis-multimers without isoform specificity involving the membrane-proximal fibronectin type III (FNIII) 1-3 and transmembrane (TM) domains and associated specifically in trans via antiparallel self-binding of the first variable immunoglobulin (Ig1) domain. We propose that such sDscam combinatorial homophilic specificity is sufficient to provide each neuron with a unique identity for self/non-self discrimination. In many respects, our results amazingly mirror those reported for the structurally unrelated vertebrate protocadherins (Pcdh) rather than for the closely related fly Dscam1. Thus, our findings blur the distinction between the neuronal self-avoidance of invertebrates and vertebrates and provide insight into the basic principles and evolution of metazoan self-avoidance and self/non-self discrimination.

1807: Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems
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Posted to bioRxiv 19 Aug 2019

Assessment of a western blot signal for the Bcnt/Cfdp1, a tentative component of Srcap chromatin remodeling complex; trial to overcome off-target problems
260 downloads biochemistry

Shintaro Iwashita, Takehiro Suzuki, Yoshimitsu Kiriyama, Naoshi Dohmae, Yoshiharu Ohka, Si-Young Song, Kentaro Nakashima

The BCNT (Bucentaur) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in fungi, fly, and chicken. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the Srcap (SNF2-Related CBP Activator Protein) chromatin remodeling complex, but little is known about its properties, partly because there are few suitable antibodies to detect the endogenous protein. We used multiple anti-Bcnt/Cfdp1 antibodies against unrelated immunogens derived from BCNT-C domain and mouse-specific N-terminal peptide. To assign western blot signals and evaluate these antibodies, we utilized a stem cell line from mutant embryos of mouse Bcnt/Cfdp1 , whose mRNA expression levels were reduced to 75% of the parental cells. In western blotting of these mutant and parental cell extracts with the anti-Bcnt/Cfdp1 antibodies, mouse Bcnt/Cfdp1 was detected as a doublet of approximately 45 kDa. LC-MS/MS analysis of the corresponding doublet for the Flag-tagged mouse Bcnt/Cfdp1 constitutively expressed in T-REx 293 cell (a HEK293 derivative) exhibited that the upper band was much more phosphorylated than the lower band and that there was preferential Ser phosphorylation in the WESF motif in the BCNT-C domain. Western blot with these validated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development in mouse and rat, which is consistent with the expression of Bcnt/Cfdp1 mRNA. This article describes the evaluation of anti-Bcnt/Cfdp1 antibodies, including a scheme to prepare a potential negative control for western blot, and discusses immune-cross reactions with off-target proteins, particularly immunoreaction probabilities.

1808: The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro
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Posted to bioRxiv 21 May 2018

The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro
260 downloads biochemistry

David G. Nickens, Cody M. Rogers, Matthew L. Bochman

Telomere length homeostasis is vital to maintaining genomic stability and is regulated by multiple factors, including telomerase activity and DNA helicases. The Saccharomyces cerevisiae Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RecQ-like helicase 4 (RECQL4), plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an in vitro primer extension assay, here we determined the effects of recombinant wild-type and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity depending on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis in vivo. We propose a model in which Hrq1 and Pif1 cooperatively contribute to telomere length homeostasis in yeast.

1809: Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking
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Posted to bioRxiv 24 Feb 2019

Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking
260 downloads biochemistry

Leslie A McCauliff, Annette Langan, Ran Li, Olga Ilnytska, Debosreeta Bose, Peter C Kahn, Judith Storch

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

1810: Native or promiscuous? Analyzing putative dimethylsulfoniopropionate lyases using a substrate proofing approach
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Posted to bioRxiv 27 Jul 2017

Native or promiscuous? Analyzing putative dimethylsulfoniopropionate lyases using a substrate proofing approach
260 downloads biochemistry

Lei Lei, Kesava Phaneendra Cherukuri, Diana Meltzer, Uria Alcolombri, Dan S. Tawfik

Enzyme promiscuity is widely spread. Foremost, within superfamilies, the native function of one enzyme is typically observed as promiscuous activity in related enzymes. The native function usually exhibits high catalytic efficiency while promiscuous activities are weak, but this is not always the case. Thus, for certain enzymes it remains questionable whether their currently known activity is native or promiscuous. Dimethylsulfoniopropionate (DMSP) is an abundant marine metabolite cleaved via beta-elimination to release dimethylsulfide (DMS). Eight different gene families have been identified as putative DMSP lyases, 5 of them belonging to the same superfamily (cupin-DLL; see the accompanying paper). Some of these enzymes exhibit very low activity, but this can be due to suboptimal folding or reaction conditions. We developed a substrate profiling approach with the aim of distinguishing native DMSP lyases from enzymes that promiscuously act as DMSP lyases. In a native DMSP lyase, relatively small changes in the structure of DMSP should induce significant activity drops. We thus profiled substrate selectivity by systematically modifying DMSP while retaining reactivity. Three enzymes that exhibit the highest activity with DMSP also exhibited high sensitivity to perturbation of DMSP structure (Alma, DddY, and DddL). The two enzymes with the weakest DMSP lyase activity also showed the highest crossreactivity (DddQ, DddP). Combined with other indications, it appears that the DMSP lyase activity of DddQ and DddP is promiscuous although their native function remains unknown. Systematic substrate profiling could help identify and assign potential DMSP lyases, and possibly applied to other enzymes.

1811: Affinity proteomic dissection of the human nuclear cap-binding-complex interactome
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Posted to bioRxiv 21 Apr 2020

Affinity proteomic dissection of the human nuclear cap-binding-complex interactome
260 downloads biochemistry

Yuhui Dou, Svetlana Kalmykova, Maria Pashkova, Mehrnoosh Oghbaie, Hua Jiang, Kelly R. Molloy, Brian T. Chait, Michael P. Rout, David Fenyö, Torben Heick Jensen, Ilya Altukhov, John LaCava

A 5', 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). The CBC has come under renewed investigative interest in recent years due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome - including the proteins SRRT (a.k.a. ARS2) and ZC3H18, and macromolecular assemblies such as the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative, non-canonical CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2-, and 3-related macromolecular assemblies, including their intersections and differences, we have applied an affinity capture-based interactome screening approach, where the experimental design and data processing have been modified and updated to identify interactome differences between targets under a range of experimental conditions, in the context of label-free quantitative mass spectrometry. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways. ### Competing Interest Statement The authors have declared no competing interest.

1812: Characterization of aroma-active compounds in delicious apples juices by gas chromatography-mass spectrometry (GC-MS), gas chromatography–olfactometry (GC-O) and odor activity value (OAV)
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Posted to bioRxiv 16 Apr 2019

Characterization of aroma-active compounds in delicious apples juices by gas chromatography-mass spectrometry (GC-MS), gas chromatography–olfactometry (GC-O) and odor activity value (OAV)
260 downloads biochemistry

Mao deshou, Hong liu, Li zhengfeng, Niu yunwei, Xiao zuobing, Zhang fengmei, Zhu jiancai

Volatile aroma compounds of delicious apple juice in three cultivars were obtained by gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O), and GC-flame photometric detection (FPD). Quantitatively, the major volatiles of the delicious apple juice were detected by GC-MS, such as esters, alcohols, aldehydes. In addition, GC-O and OAV were used to determine the aroma-active compounds in fruit. Amongst these compounds, ethyl 2-methylbutanoate (47-229), butyl 2-methylbutanoate (8-208), (E)-2-hexenal (25-120), butyl propanoate (14-54), methyl 2-methylbutanoate (28-41), ethyl hexanoate (4-32), ethyl butanoate (5-17) showed high OAVs in three delicious apple juices, which contributed greatly to the aroma of delicious apple juice. Beside those compounds, methanethiol (OAV: 1.1-1.6), dimethyl sulfide (OAV: 2.5-3.6), methional (OAV: 4.2-11.7) and 2-(methylthio)ethanol (OAV: 1.2-1.9) also presented relatively high OAVs. Finally, four compounds (ethyl 2-methylbutanoate, ethyl octanoate, ethyl butanoate and ethyl hexanoate) were selected to investigate the possible interactions occurring in the delicious apple juice. The resultants demonstrated that those aroma volatile compounds can decrease threshold of the solution to dissimilar degrees before and after their addition.

1813: The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair
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Posted to bioRxiv 18 Sep 2019

The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair
260 downloads biochemistry

Cody M. Rogers, Chun-Ying Lee, Samuel Parkins, Nicholas J. Buehler, Sabine Wenzel, Francisco Martínez-Márquez, Yuichiro Takagi, Sua Myong, Matthew L. Bochman

DNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae , a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2-mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Pso2 alone has meagre translesion nuclease activity on an ICL-containing substrate, but digestion through the lesion dramatically increases in the presence of Hrq1. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 interacts with Pso2, likely through their N-termini. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions. ### Competing Interest Statement The authors have declared no competing interest.

1814: Structural Basis for CAL1-Mediated Centromere Maintenance
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Posted to bioRxiv 02 Aug 2019

Structural Basis for CAL1-Mediated Centromere Maintenance
260 downloads biochemistry

Bethan Medina-Pritchard, Vasiliki Lazou, Juan Zou, Olwyn Byron, Juri Rappsilber, Patrick Heun, A. Arockia Jeyaprakash

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting is achieved by HJURP and the Mis18 complex, respectively. Here, using crystal structures, we show how Drosophila CAL1, an evolutionarily distinct CENP-A chaperone, targets CENP-A to the centromere receptor CENP-C without the requirement of the Mis18 complex: while the N-terminal CAL1 fragment (CAL1 1-160) wraps around CENP-A/H4 through multiple physical contacts, the C-terminal CAL1 fragment (CAL1 893-914) directly binds CENP-C cupin dimer. Our work shows CAL1, though divergent at the primary structure, employs evolutionarily conserved and adaptive structural principles to recognise CENP-A/H4 and CENP-C providing insights into the minimalistic principles underlying centromere maintenance.

1815: Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail
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Posted to bioRxiv 29 Mar 2020

Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail
259 downloads biochemistry

Yuxia Zhang, Michio Inoue, Akihisa Tsutsumi, Satoshi Watanabe, Tomohiro Nishizawa, Kazuhiro Nagata, Masahide Kikkawa, Kenji Inaba

SERCA2b is a Ca2+-ATPase that pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-EM structures of human SERCA2b in E1·2Ca2+-AMPPCP and E2-BeF3- states at 2.9 and 2.8 Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7, and approaches the luminal loop flanked by TM7 and TM8. Upon deletion of the LE, the cytosolic- and TM-domain arrangement of SERCA2b resembled that of SERCA1a, resulting in multiple conformations. The LE regulates the conformational transition between the E1·2Ca2+-ATP and E2P states, explaining the different kinetic properties of SERCA2b from other isoforms lacking the LE.

1816: Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells
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Posted to bioRxiv 07 Oct 2019

Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells
259 downloads biochemistry

Robert P. Sparks, Andres S. Arango, Zachary L Aboff, Jermaine L Jenkins, Wayne C Guida, Emad Tajkhorshid, Charles E Sparks, Janet D Sparks, Rutilio A. Fratti

Sortilin regulates hepatic exocytosis and endocytosis of ApoB containing lipoproteins (ApoB-Lp) and mediates the secretion of the subtilase PCSK9. To elucidate connections between these pathways, we previously identified a small molecule (cpd984) that binds to a non-canonical site on Sortilin. In hepatic cells cpd984 augments ApoB-Lp secretion, increases cellular PCSK9 levels, and reduces LDLR expression indicative of reduced secretion of PCSK9. We have shown that insulin-induced ApoB-Lp degradation occurs through Vps34-dependent autophagy. Here we show that the specific Vps34 inhibitor PIK-III enhances ApoB-100 secretion, reducing cellular levels of PCSK9 and Sortilin resulting in reduced LDLR expression, which implicates a role for autophagy in PCSK9 secretion. Results suggest that Sortilin is central to both PCSK9 and ApoB-100 secretion. Finally, we found that cpd984 in yeast blocks CPY secretion while increasing vacuolar homotypic fusion in a Vps10-dependent manner, indicating an evolutionarily conserved mechanism required for lysosomal protease trafficking.

1817: The flexible N-terminus of BchL protects its [4Fe-4S] cluster in oxygenic environments and autoinhibits activity
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Posted to bioRxiv 12 Nov 2019

The flexible N-terminus of BchL protects its [4Fe-4S] cluster in oxygenic environments and autoinhibits activity
259 downloads biochemistry

Elliot Corless, Syed Muhammad Saad Imran, Maxwell B Watkins, Sofia Origanti, John-Paul Bacik, Robert Kitelinger, Mark 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 relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one de-fective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

1818: Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ
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Posted to bioRxiv 20 Aug 2019

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ
259 downloads biochemistry

Alan Sulpizio, Marena E. Minelli, Min Wan, Paul D. Burrowes, Xiaochun Wu, Ethan Sanford, Jung-Ho Shin, Byron Williams, Michael Goldberg, Marcus Smolka, Yuxin Mao

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

1819: Alternative splicing of bicistronic MOCS1 defines a novel mitochondrial protein maturation mechanism
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Posted to bioRxiv 27 Sep 2018

Alternative splicing of bicistronic MOCS1 defines a novel mitochondrial protein maturation mechanism
258 downloads biochemistry

Simon Julius Mayr, Juliane Röper, Geunter Schwarz

Molybdenum cofactor biosynthesis is a conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires bicsistronic MOCS1. Alternative splicing of MOCS1 in exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I-III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces two-domain MOCS1AB proteins. Here, we report and characterize the mitochondrial translocation of alternatively spliced MOCS1 proteins. While MOCS1A requires exon 1a for mitochondrial translocation, MOCS1AB variants target to mitochondria via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB undergoes proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion we found that MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import, where both proteins collectively catalyze cPMP biosynthesis. MOCS1 protein maturation provides a novel mechanism of alternative splicing ensuring the coordinated targeting of two functionally related mitochondrial proteins encoded by a single gene.

1820: A Dry Method For Preserving Tear Samples
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Posted to bioRxiv 26 Apr 2017

A Dry Method For Preserving Tear Samples
258 downloads biochemistry

Weiwei Qin, Chan Zhao, Linpei Zhang, Ting Wang, Youhe Gao

Tears covering the ocular surface is an important bio-fluid containing thousands of molecules, including proteins, lipids, metabolites, nucleic acids, and electrolytes. Tears are valuable resources for biomarker research of ocular and even systemic diseases. For application in biomarker studies, tear samples should ideally be stored using a simple, low-cost, and efficient method along with the patient's medical records. For this purpose, we developed a novel Schirmer's strip-based dry method that allows for storage of tear samples in vacuum bags at room temperature. Using this method, tear protein patterns can also be preserved. Liquid chromatography-mass spectrometry/mass spectrometry analysis of proteins recovered from the dry method and traditional wet method showed no significant difference. This dry method facilitates sample transportation and enables the storage of tear samples on a large scale, increasing the availability of samples for studying biomarker of diseases in tears.

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