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bioRxiv2019

In vitro and in silico identification of the mechanism of interaction of antimalarial drug - artemisinin with human serum albumin and genomic DNA

Artemisinins are secondary metabolites of the medicinal plant Artemisia annua, which has been traditionally used in Chinese medicine. Artemisinins have anti-inflammatory, anticarcinogenic, immunomodulatory, antimicrobial, anthelmintic, antiviral, antioxidant, and other properties. Our preliminary reverse virtual screening demonstrated that the ligand-binding domain of the human glucocorticoid receptor (LBD of hGR) is the optimal target for artemisinin. At the same time, the binding sites for artemisinin with the ligand-binding domain of the human glucocorticoid receptor coincide with those of dexamethasone. However, the pharmacokinetics, pharmacodynamics, and exact molecular targets and mechanisms of action of artemisinin are not well known. In this work, the interaction of artemisinin with human serum albumin (HSA) was studied both in vitro and in silico. The results indicate that artemisinin leads to a decrease in optical absorption and quenching of fluorescence by a static mechanism, which is similar to the effect of dexamethasone. Artemisinin interacts with Drug site I on HSA and forms a hydrogen bond with arginine 218. Retardation of the genomic DNA of sarcoma S-180 cells show that artemisinin does not interact directly with DNA. On the basis of the obtained data, we proposed a hypothetical scheme of the mechanisms of action of artemisinin.\n\nHighlightsO_LIArtemisinin quenches the fluorescence of HSA by a static mechanism.\nC_LIO_LIArtemisinin quenches fluorescence of tryptophan.\nC_LIO_LIThe optimized HSA structure was obtained through molecular dynamics simulations.\nC_LIO_LIArtemisinin binds with HSA in Drug site I and forms a hydrogen bond with Arg218.\nC_LIO_LIDexamethasone binds with HSA in Drug site I and forms hydrogen bonds with Arg218, Arg222 and Va1343.\nC_LIO_LIA hypothetical scheme of the mechanism of action of Artemisinin was proposed.\nC_LI\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC=\"FIGDIR/small/519710_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@cd144forg.highwire.dtl.DTLVardef@1f05519org.highwire.dtl.DTLVardef@16313aborg.highwire.dtl.DTLVardef@1a078ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira. The kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane components contain several virulence factors that play important roles in the pathogenesis of leptospirosis. Among them, OmpA-like protein Loa22 is essential for leptospiral virulence. However, the pathogenic mechanisms of tubulointerstitial nephritis involving this virulence factor are still unclear and need further investigation. In this study, pull-down assays suggested that Toll-like receptor 2 (TLR2) proteins interacted with Loa22 from Leptospira outer membrane extractions. Combination of Atomic force microscopy (AFM) and side-directed mutagenesis suggested that Loa22 exhibited high affinity for Leptospira peptidoglycan (LPGN) and the residues of Loa22 were involved in LPGN interaction. Mutation of two key residues within the OmpA-like domain of Loa22, Asp122 and Arg143, significantly attenuated their relative affinities for LPGN indicating that these two residues were responsible for LPGN binding. Thus Loa22 OmpA domain was responsible for interacting with LPGN and the two indicated residues may participate in binding to LPGN. Recombinant Loa22 (rLoa22) protein was further complexed with LPGN and incubated with HEK293-TLR2 cells to monitor inflammatory responses. Inflammatory responses were provoked by rLoa22-LPGN complexes, but not rLoa22 alone, involved CXCL8/IL8, hCCL2/MCP-1, and hTNF- activation. Confocal microscopy further identified the co-localization of Loa22-LPGN complexes and TLR2 receptors on HEK293-TLR2 cell surface. The affinity between Loa22-LPGN complexes and TLR2 were further confirmed and measured by AFM and ELISA. Downstream signals from TLR2 including p38, ERK, and JNK were observed by western blotting induced by Loa22-LPGN complexes. In summary, this study identified LPGN in leptospira mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. Interactions between Loa22-LPGN-TLR2 reveal a novel binding mechanism for the innate immune system and infection induced by leptospira.\n\nAuthor summaryLeptospirosis is one of the most overlooked zoonotic diseases caused by pathogenic Leptospira in warm and humid regions worldwide. With the infection by Leptospira, many organs are invaded and can result in multiple-organ failure (Weils syndrome). Kidney is the major organ infected by pathogenic Leptospira, which would manifest as tubulointerstitial nephritis. In this study, we focused on the outer membrane lipoprotein Loa22 (Leptospiral OmpA-like domain 22) from pathogenic Leptospira which triggers inflammatory responses on renal tubular cell. Protein domain prediction indicated that Loa22 contains an important domain termed OmpA-like domain and the function of this domain is peptidoglycan (PGN) binding. From sequence alignments of Loa22 with other OmpA proteins, two important amino acids, Asp122 and Arg143, were found to be highly conserved. The role of the two conserved residues in AbOmpA (OmpA protein in A. baumannii) and Pal (peptidoglycan-associated lipoprotein in E. coli) proteins are important for PGN binding. These two residues in Loa22 were altered by site-directed mutagenesis to obtain D122A and R143A variants. In pull-down and AFM analysis, the binding capacities of Loa22 variants to Leptospira PGN (LPGN) were significantly decreased as compared to rLoa22WT, indicating that the two residues are involved in LPGN binding. Furthermore, recombinant Loa22 and its variants in the absence or presence of LPGN, were incubated with HEK293-TLR2 cells, to confirm the role of LPGN in triggering inflammatory responses involving CXCL8/IL8, hCCL2/MCP-1, and hTNF-. These factors are involved in downstream signaling of inflammatory responses through Toll-like receptor 2 (TLR2). In addition, confocal microscopy was employed to observe the co-localization of Loa22-LPGN complexes and TLR2 receptors on HEK293-TLR2 cell surfaces. Finally, the interaction forces between rTLR2 and rLoa22-LPGN complexes were measured by AFM and ELISA to conclude the necessary role of LPGN in rLoa22-TLR2 complex formation. In summary, these results demonstrate that the interaction of Loa22 protein with the important cell wall component, PGN, concomitantly triggered inflammatory responses of host cells through interaction with TLR2.

biochemistry↗

bioRxiv2019

Discovery of a redox-thiol switch regulating cellular energy metabolism

Previously, we reported that increased synthesis of the gas hydrogen sulfide (H2S) during the Integrated Stress Response (ISR) induced proteome-wide cysteine-sulfhydration with the predominant modified pathway being enzymes of cellular energy metabolism (Gao, et al. 2015). Using pancreatic beta cells and quantitative proteomics in this study, we identified a Redox Thiol Switch from S-glutathionylation to S-sulfhydration and we named it, RTSGS. About half of the identified proteins are involved in energy metabolism, and one novel target was the mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) whose catalytic Cys306was targeted by both modifications. The enzymatic activity of PCK2 was inhibited by S-glutathionylation, and this inhibition was largely reversed by S-sulfhydration. S-sulfhydration also reversed the S-glutathionylation-mediated inhibition of glucose flux, indicating a broad metabolic significance. We propose that a Redox Thiol Switch from S-glutathionylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

biochemistry↗

bioRxiv2019

Chromokinesins NOD and KID Use Distinct ATPase Mechanisms and Microtubule Interactions to Perform a Similar Function

Chromokinesins NOD and KID have similar DNA binding domains and functions during cell division, while their motor domain sequences show significant variations. It has been unclear whether these motors have similar structure, chemistry, and microtubule interactions necessary to follow a similar mechanism of force mediation. We used biochemical rate measurements, cosedimentation, and structural analysis to investigate the ATPase mechanisms of the NOD and KID core domains. These experiments and analysis revealed that NOD and KID have different ATPase mechanisms, microtubule interactions, and catalytic domain structures. The ATPase cycles of NOD and KID have different rate limiting steps. The ATPase rate of NOD was robustly stimulated by microtubules albeit its microtubule affinity was weakened in all nucleotide bound states. KID bound microtubules tightly in all nucleotide states and remained associated with the microtubule for more than 100 cycles of ATP hydrolysis before dissociating. The structure of KID was most similar to conventional kinesin (KIF5). Key differences in the microtubule binding region and allosteric communication pathway between KID and NOD are consistent with our biochemical data. Our results support the model that NOD and KID utilize distinct mechanistic pathways to achieve the same function during cell division.

biochemistry↗

bioRxiv2019

Stimulation of Gαq Promotes Stress Granule Formation

During adverse conditions, mammalian cells regulate protein production by sequestering the translational machinery in membrane-less organelles known as stress granules. Here, we found that activation of the G protein subunit Gq promoted the formation of particles that contained stress granule proteins through a mechanism linked to the presence of phospholipase C{beta}1 (PLC{beta}1) in the cytosol. In experiments with PC12 and A10 cells, we showed that under basal conditions, cytosolic PLC{beta}1 bound to stress granule associated proteins, including PABPC1, eIF5A, and Ago2. Knockdown of cytosolic PLC{beta}1 with siRNA or promoting its relocalization to the plasma membrane by activating Gq resulted in the formation of particles containing the stress granule markers, PABPC1, G3BP1, and Ago2. Our studies showed that the composition of these particles resemble those formed under osmotic stress and are distinct from those formed by other stresses. Our results fit a simple thermodynamic model in which cytosolic PLC{beta}1 solubilizes stress granule proteins such that its movement to activated Gq releases these particles to enable the formation of stress granules. Together, our data are suggestive of a link between Gq-coupled signals and protein translation through stress granule formation.

biochemistry↗

bioRxiv2019

A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure and function by forming transient intermediate folds. This is especially true for transcriptional riboswitches in the which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways and the mechanisms that enable transit between them remain largely unknown. Here we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate a regulatory RNA strand displacement reaction and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for ZTP riboswitch antitermination and suggest general strategies by which nascent RNA molecules can navigate cotranscriptional folding pathways efficiently.

biochemistry↗

bioRxiv2019

Fibril formation rewires interactome of the Alzheimer protein Tau by π-stacking

Aggregation of the Tau protein defines progression of neurodegenerative diseases, including Alzheimers Disease. Tau assembles into oligomers and fibrils. The molecular basis of their toxicity is poorly understood. Here we show that {pi}-stacking by Arginine side chains rewires the interactome of Tau upon aggregation. Oligomeric nano-aggregates scavenge the COPI complex, fibrils attract proteins involved in microtubule binding, RNA binding and phosphorylation. The aberrant interactors have disordered regions with unusual sequence features. Arginines are crucial to initiate such aberrant interactions. Remarkably, substitution of Arginines by Lysines abolishes scavenging, which indicates a key role for the pi-stacking of the Arginine side chain. The molecular chaperone Hsp90 tames such re-arrangements, which suggests that the natural protein quality control system can suppress aberrant interactions. Together, our data present a molecular mode of action for derailment of protein-protein interaction in neurodegeneration.\n\nHIGHLIGHTSO_LITau fibrils act as fishing net for proteins.\nC_LIO_LITau fibrils attract specific protein families associated with Alzheimer.\nC_LIO_LI{pi}-stacking by Arginines key for aberrant binding to Tau fibrils\nC_LIO_LIThe Hsp90 chaperone stalls fibril growth and alters interactome\nC_LI

biochemistry↗

bioRxiv2019

A widespread Xrn1-resistant RNA motif composed of two short hairpins

Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3 untranslated region of several beny-and cucumovirus RNAs harbors a so-called coremin motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence, to deduce putative structural features responsible for Xrn1-stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5 proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 9-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn-stalling motifs are more widespread among RNA viruses than previously realized.

biochemistry↗

bioRxiv2019

X-ray structures of two active secreted Bacteroides thetaiotaomicron C11 proteases in complex with peptide-based inhibitors

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed \"thetapain\" and \"iotapain\", undergo in trans self-maturation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.

biochemistry↗

bioRxiv2019

Mechanistic Analyses of Supercoiling Behaviors of DNA in Nucleosomes and Chromatins

Besides those in 146-base pair nucleosome core particle DNA, supercoils have been known to be present in 10-base pair arm DNA segments and naked linker DNA segments. The interacting patterns among histone octamers, histone H1, 10-base pair arm DNA segments and linker DNA have, however, not yet been elucidated. In the current report, we examine correlations among constituents of nucleosomes from the mechanistic perspectives and present molecular pathways for elucidating supercoiling behaviors of their component DNA sequences. It is our hope that our new analyses could serve as incentives to further clarify correlations between histones and DNA in the dynamic structures of chromatins in the future.

biochemistry↗

bioRxiv2019

Development of Triazoles based on AZT and their Anti-Viral Activity Against HIV-1

We report herein a set of 3-azido-3-deoxythymidine (AZT) derivatives based on triazoles and triazolium salts for HIV1 infection. Compounds were tested with {square}using HIV1 pre-exposure prophylaxis experimental model. All compounds were able to decrease infection and two of them were able to clear almost all the infection, suggesting that these drugs could play an important role in pre-exposure prophylaxis therapies.

biochemistry↗

bioRxiv2019

Compartmentalized Biosynthesis of Mycophenolic Acid

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic in human history and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, its biosynthetic mechanisms have remained a long-standing mystery. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and {beta}-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB as the long-sought key enzyme responsible for the oxidative cleavage of sesquiterpene side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC and O-methyltransferase MpaG, the Golgi apparatus-associated prenyltransferase MpaA, the endoplasmic reticulum-bound oxygenase MpaB and P450-hydrolase fusion enzyme MpaDE, and the peroxisomal acyl-CoA hydrolase MpaH. The whole pathway is elegantly co-mediated by these compartmentalized enzymes, together with the peroxisomal {beta}-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic pathway, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.\n\nSignificance StatementHere we elucidate the full biosynthetic pathway of the fungal natural product mycophenolic acid (MPA), which represents an unsolved mystery for decades. Besides the intriguing enzymatic mechanisms, we reveal that the MPA biosynthetic enzymes are elegantly compartmentalized; and the subcellular localization of the acyl-CoA hydrolase MpaH in peroxisomes is required for the unique cooperation between biosynthetic and {beta}-oxidation catabolism machineries. This work highlights the importance of a cell biology perspective for understanding the unexplored organelle-associated essential catalytic mechanisms in natural product biosynthesis of fungi and other higher organisms. The insights provided by our work will also benefit future efforts for both industrial strain improvement and novel drug development.

biochemistry↗

bioRxiv2019

Integrin cytoplasmic domain and pITAM compete for spleen tyrosine kinase binding

In hematopoietic tissues cell-cell communication involves immunoreceptors and specialized cell adhesion receptors that both mediate intracellular signals. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in the downstream signaling of both immunoreceptors tyrosine activation motif (ITAM) receptors and integrin family cell adhesion receptors. Both phosphorylated ITAM (pITAM) and integrins bind to the regulatory domain of Syk composed of two Src homology 2 (SH2) domains. The interaction with pITAM is mediated by binding of a specific phosphotyrosine to each of the SH2 domains, leading to conformational changes and Syk kinase activation. Integrins bind to the interdomain A segment between the two SH2 domains and to the N-terminal SH2 domain, but the detailed binding site is not known. In order to map the binding site, we performed NMR titration experiments. We found that integrin cytoplasmic domain peptide induced chemical shift changes near the IA segment and at the phosphotyrosine binding site of the N-terminal SH2 domain of Syk. These changes were distinct, but partially overlapping with those induced by pITAM peptide. We were also able to show that pITAM peptide inhibited integrin binding to Syk regulatory domain. These results suggest that ITAM receptors and integrins cannot bind simultaneously to Syk, but provide two distinct routes for Syk activation.

biochemistry↗

bioRxiv2019

Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis

Summary ParagraphProtein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzymes conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at Gly150 that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.

biochemistry↗

bioRxiv2019

Chaperone-mediated ordered assembly of the SAGA transcription complex

Transcription initiation involves the coordinated activities of large multimeric complexes that are organized into functional modules. Little is known about the mechanisms and pathways that govern their assembly from individual components. We report here several principles governing the assembly of the highly conserved SAGA and NuA4 co-activator complexes. Using fission yeast, which contain two functionally non-redundant paralogs of the shared Tra1 subunit, we demonstrate that Tra1 contributes to scaffolding the entire NuA4 complex. In contrast, within SAGA, Tra1 specifically promotes the incorporation of the de-ubiquitination module (DUB), defining an ordered assembly pathway. Biochemical and functional analyses elucidated the mechanism by which Tra1 assemble differentially into SAGA or NuA4 and identified a small, conserved region of Spt20 that is both necessary and sufficient to anchor Tra1 within SAGA. Finally, we establish that Hsp90 and its cochaperone TTT are required for Tra1 de novo incorporation into both SAGA and NuA4, indicating that Tra1, a pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the de novo assembly of transcriptional complexes through ordered pathways and reveals the contribution of dedicated chaperones to this process.

biochemistry↗

bioRxiv2019

Structural Insights into GluK3-kainate Receptor Desensitization and Recovery

GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play key role in regulation of synaptic transmission. For better understanding of structural changes that underlie receptor recovery from desensitized state, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. We show that receptor recovery from desensitization requires major rearrangements of the ligand binding domains (LBD) while the amino terminal (ATD) and transmembrane domains remain virtually unaltered. While, the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a partially \"recovered\" state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using these structures as guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. Mutational analysis also identifies putative N-glycan interacting residues. These results provide a molecular framework for understanding gating properties unique to GluK3 and identify role of N-linked glycosylation in their modulation.

biochemistry↗

bioRxiv2019

The large loop-8 insert in Saccharomyces cerevisiae kinesin-5 Cin8 is necessary and sufficient to promote noncanonical microtubule interactions

Saccharomyces cerevisiae kinesin-5 Cin8 displays unconventional biochemical behavior including bidirectional motility and ability to bind multiple motor domains per {beta} tubulin dimer in the microtubule lattice. Previous research suggested that a large loop-8 insert near the microtubule binding interface of Cin8 was critical for its noncanonical microtubule binding behavior. Here we utilized mutagenesis, thermodynamic, and kinetic assays to further understand the mechanism for how this loop-8 insert promotes super-stoichiometric microtubule binding in Cin8. This loop-8 insert that interrupts the conserved {beta}5a/b hairpin was swapped between Cin8, Eg5 (KIF11, a human kinesin-5) and Kip1 (another S. cerevisiae kinesin-5). Cin8 with the loop-8 insert from Eg5 (Cin8-EL8) binds one motor per tubulin dimer, whereas Eg5 with the loop-8 insert from Cin8 (Eg5-CL8) binds approximately 2-4 motors per tubulin dimer. Eg5-CL8 bound the canonical and noncanonical sites on the microtubule lattice with weakened oligomerization between motors, while Cin8-EL8 showed only canonical site binding. These results demonstrate that the large loop-8 insert in Cin8 is necessary and sufficient to promote noncanonical microtubule binding behavior.

biochemistry↗

bioRxiv2019

Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called UPR (Unfolded Protein Response) that functions to maintain ER (endoplasmic reticulum) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification (PTM) called Fic-mediated Adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an AMP (adenosine monophosphate) to the ER chaperone, BiP, to alter the cells UPR-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE - alpha-Synuclein (Syn), a presynaptic protein involved in Parkinsons disease (PD). Aggregated Syn has been shown to induce ER stress and elicit neurotoxicity in PD models. We show that HYPE adenylylates Syn and reduces phenotypes associated with Syn aggregation in vitro, suggesting a possible mechanism by which cells cope with Syn toxicity.\n\nHIGHLIGHTSO_LIAggregated forms of the presynaptic protein Syn cause neurotoxicity and induce ER stress in cellular and animal models of Parkinsons disease.\nC_LIO_LIWe have identified Syn as a novel target for the human Fic protein, HYPE, a key regulator of ER homeostasis.\nC_LIO_LIHYPE adenylylates Syn and reduces the aggregation of recombinant Syn\nC_LIO_LIFic-mediated adenylylation/AMPylation is a possible mechanism by which cells cope with Syn toxicity.\nC_LI\n\nGraphic Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗