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<title>bioRxiv Subject Collection: Biochemistry</title>
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This feed contains articles for bioRxiv Subject Collection "Biochemistry"
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<link>https://www.biorxiv.org</link>
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<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.08.723602v1?rss=1">
<title>
<![CDATA[
Molecular architecture of meiotic pro-crossover factor HEI10 reveals coupling of higher-order assembly and ubiquitin chain formation 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.08.723602v1?rss=1
</link>
<description><![CDATA[
In meiosis, crossovers between homologous chromosomes generate genetic diversity and are required for accurate chromosome segregation, ensuring fertility. In mammals, HEI10 is one of three pro-crossover RING-domain factors implicated in protein modification by ubiquitin and/or SUMO and characterised by their dynamic accumulation at future crossover sites. However, the molecular architecture and enzymatic activity of mammalian HEI10 have remained unknown. Here, we show that human HEI10 has E3-ubiquitin ligase activity that depends on its higher-order assembly. We report the crystal structure of the HEI10 core, revealing how a 29-nm rod-like tetramer is formed through head-to-head association of two coiled-coil dimers that results in clustering of four RING domains around the molecular centre. HEI10 tetramers self-assemble through RING, coiled-coil, and C-terminal interfaces into fibrous and spherical higher-order structures. Structure-guided mutants show that higher-order assembly is required for HEI10 to catalyse K63-linked ubiquitin chain formation in vitro, with the most active species likely corresponding to a loose, non-fibrous network of assembled HEI10 molecules. Arabidopsis thaliana HEI10 retains the tetrameric core and higher-order assembly behaviour, suggesting a conserved principle of HEI10 function.
]]></description>
<dc:creator><![CDATA[ Milburn, A. E., Kulkami, D. S., Espejo-Serrano, C., Pachon-Penalba, M., Williams, M. E., Nicol, J. P. O., Debilio, S., Gurusaran, M., Dunce, J. M., Adams, I. R., McClurg, U. L., Hunter, N., Davies, O. R. ]]></dc:creator>
<dc:date>2026-05-10</dc:date>
<dc:identifier>doi:10.64898/2026.05.08.723602</dc:identifier>
<dc:title><![CDATA[Molecular architecture of meiotic pro-crossover factor HEI10 reveals coupling of higher-order assembly and ubiquitin chain formation]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-10</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723680v1?rss=1">
<title>
<![CDATA[
Molecular basis for the role of Ripr in Plasmodium falciparum invasion of human erythrocytes 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723680v1?rss=1
</link>
<description><![CDATA[
Plasmodium falciparum causes the majority of severe malaria, and merozoite invasion of erythrocytes is a vulnerable, antibody-accessible step of the blood-stage cycle. PfRipr is an essential component of the PCRCR invasion complex, yet the structural basis for antibody-mediated neutralisation remains unclear. Here, we map inhibitory and non-inhibitory epitopes across PfRipr and show that all potent inhibitors localise to the tail region (EGF6-8). Crystal structures reveal that inhibitory antibodies restrict the flexibility surrounding EGF7. Indeed, EGF7 buried surface area correlates strongly with inhibitory potency, identifying this domain as the principal invasion inhibitory determinant. Pairwise antibody combinations revealed unexpected synergy, with non-inhibitory mAbs potentiating anti-Rh5 activity. Conditional deletion, sequence replacement or positional swapping of EGF6-8 abolished invasion, demonstrating that both sequence and spatial arrangement are indispensable. These data define EGF7 as a conserved, functionally essential vulnerability and provide a blueprint for rational EGF6-8 immunogen design capable of eliciting P. falciparum strain-transcending protection against blood-stage malaria.
]]></description>
<dc:creator><![CDATA[ Xiao, X., Zhou, Q., Marapana, D. F., McLean, T. C., Chan, R. W. B., Shakeel, S., Leis, A., Lim, P. S., Jung, N. C., Naung, M., Lopaticki, S., Dixon, M. W. A., Barry, A. E., Cowman, A. F., Scally, S. W. ]]></dc:creator>
<dc:date>2026-05-09</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723680</dc:identifier>
<dc:title><![CDATA[Molecular basis for the role of Ripr in Plasmodium falciparum invasion of human erythrocytes]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-09</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723538v1?rss=1">
<title>
<![CDATA[
A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723538v1?rss=1
</link>
<description><![CDATA[
The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.
]]></description>
<dc:creator><![CDATA[ Kaushik, V., Sanjayan, V., Mattice, J., Tokmina-Lukaszewska, M., Toerner, R., Chadda, R., Kashyap, R., Vayyeti, A., Roy, P., Mehl, R., Cooley, R. B., Por Sigurdsson, S., Dastvan, R., Arthanari, H., Bothner, B., Origanti, S. S., Antony, E. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723538</dc:identifier>
<dc:title><![CDATA[A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723605v1?rss=1">
<title>
<![CDATA[
Localized heme sensing through a ternary molecular glue 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723605v1?rss=1
</link>
<description><![CDATA[
Molecular glues are an emerging class of therapeutics that stabilize binary interactions and thereby rewire disease-relevant protein networks. Whether glues can integrate additional information to orchestrate signaling beyond initial complex formation is unknown. Here, we report that cells use an endogenous glue strategy to sense heme, an essential metabolite with deleterious pro-oxidant properties. Distinct from other glues, heme bridges three polypeptides to trigger degradation of the transcriptional repressor BACH1 through cytoplasmic, but not mitochondrial, CUL2FEM1B. This mechanism allows cells to eliminate toxic heme in the cytoplasm by inducing expression of the heme-degrading oxygenase HMOX1, yet ignore mitochondrial heme destined for function in the electron transport chain. While protective in healthy cells, ternary glue signaling creates a therapeutic vulnerability for Acute Myeloid Leukemias dependent on high rates of ETC assembly. Molecular glues can therefore drive assembly of higher-order complexes to establish localized signaling, which offers unexplored opportunities for induced proximity therapeutics.
]]></description>
<dc:creator><![CDATA[ Heider, M., Hipp, C., Yang, Z., Xiao, H., Beschauner, T., Wehri, E., Walter, W., Sherriff, R., Chandrasekhar, S., Haferlach, T., Schaletzky, J., Rape, M. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723605</dc:identifier>
<dc:title><![CDATA[Localized heme sensing through a ternary molecular glue]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723500v1?rss=1">
<title>
<![CDATA[
A de novo CO2 Reductase Featuring a Cysteine-Ligated Cobalt Porphyrin Cofactor 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723500v1?rss=1
</link>
<description><![CDATA[
Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.
]]></description>
<dc:creator><![CDATA[ Radley, E., Andrews, A., Kalvet, I., Deng, Y., Levy, C., Ortmayer, M., Heyes, D., Megarity, C., Nunez-Franco, R., Hutton, A., Lu, Y., Baker, D., Green, A. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723500</dc:identifier>
<dc:title><![CDATA[A de novo CO2 Reductase Featuring a Cysteine-Ligated Cobalt Porphyrin Cofactor]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722967v1?rss=1">
<title>
<![CDATA[
Elucidation of the anti-inflammatory mechanism of isoliquiritigenin from Glycyrrhiza uralensis using activity-based protein profiling 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722967v1?rss=1
</link>
<description><![CDATA[
Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.
]]></description>
<dc:creator><![CDATA[ Sakai, H., Chung, M. H., Nakaya, T., Ohbuchi, K., Isobe, Y., Arita, M., Tsumagari, K., Imami, K., Hirokawa, T., Tsugawa, H. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722967</dc:identifier>
<dc:title><![CDATA[Elucidation of the anti-inflammatory mechanism of isoliquiritigenin from Glycyrrhiza uralensis using activity-based protein profiling]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.723150v1?rss=1">
<title>
<![CDATA[
The development of ToF-SIMS for in-situ glycosaminoglycan analysis 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.723150v1?rss=1
</link>
<description><![CDATA[
lycosaminoglycans (GAGs) are linear polysaccharides with essential roles in a myriad of biological processes. Despite their biological importance, methods to determine both spatial and compositional information is limited. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides spatially resolved compositional information of biological molecules without enzymatic digestion or label incorporation, enabling unbiased analysis independent of enzyme or label selectivity, overcoming many current limitations in GAG analysis. Here, we present the identification and validation of GAG discriminatory ions from biological samples by comparison of spectra from purified GAGs and cells with genetically modified GAG biosynthetic pathways. Ions discriminatory of specific GAG sub-families are identified and related to GAG structural components. The analysis is applied to human induced pluripotent stem cells engineered to lack heparan sulphate (HS), where compensatory changes in GAG display that link to function were observed. Furthermore, the broad applicability and spatial resolution of the technique is highlighted through detection of a disease-induced reduction in HS within the individual glomeruli of diabetic mice.
]]></description>
<dc:creator><![CDATA[ Milne, L. K., Thompson, J. L., Ramnath, R. D., Satchell, S., Miller, R. L., Kjellen, L., Arkill, K. P., Merry, C. L. R., Hook, A. L. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.723150</dc:identifier>
<dc:title><![CDATA[The development of ToF-SIMS for in-situ glycosaminoglycan analysis]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723540v1?rss=1">
<title>
<![CDATA[
More than an attachment module: covalent inhibitor warheads influence BTK dynamics and function. 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723540v1?rss=1
</link>
<description><![CDATA[
Covalent inhibitors are rapidly becoming the standard of care for treatment of a range of disease states. Covalent inhibitors bind irreversibly to their target using a reactive electrophile (or warhead). Acrylamide and 2-butynamide are the most commonly used cysteine targeting electrophiles. These warheads are chosen for their efficient and selective modification of the protein and are presumed to be otherwise functionally inert. Using a panel of BTK covalent inhibitors (Tirabrutinib, Acalabrutinib, Ibrutinib and Zanubrutinib), we show that the 2-butynamide warhead on Tirabrutinib and Acalabrutinib, unlike the acrylamide warhead on Ibrutinib and Zanubrutinib, induces conformational heterogeneity in key regions required for BTK signaling. Tirabrutinib or Acalabrutinib bound BTK adopt multiple conformational states that are in dynamic exchange, show increased binding to the substrate PLCgamma; and are less effective at inhibiting PLCgamma; signaling when compared to Ibrutinib. Swapping only the warheads between Tirabrutinib and Ibrutinib leads to a corresponding switch in BTK dynamics and inhibitor efficacy. The unanticipated warhead-specific allosteric effects raise interesting possibilities regarding inhibitor-specific mechanisms of resistance.
]]></description>
<dc:creator><![CDATA[ Joseph, R. E., Britton, R. G., Lin, D. Y.-w., Roche, J., Purslow, J. A., Fulton, D. B., Fukasem, P., Gleeson, M. P., Dyer, M. J. S., Wales, T. E., Andreotti, A. H. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723540</dc:identifier>
<dc:title><![CDATA[More than an attachment module: covalent inhibitor warheads influence BTK dynamics and function.]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723505v1?rss=1">
<title>
<![CDATA[
Hydrophobic pocket engineering of arylmalonate decarboxylase expands its substrate scope towards the synthesis of the (R)-enantiomers of sterically hindered carboxylic acids 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723505v1?rss=1
</link>
<description><![CDATA[
Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of alpha-aryl and alpha-alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity
]]></description>
<dc:creator><![CDATA[ van der Pol, E., Krammer, L.-M., Eder, J., Gross, D., Fischer, R., Miyamoto, K., Breinbauer, R., Kourist, R. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723505</dc:identifier>
<dc:title><![CDATA[Hydrophobic pocket engineering of arylmalonate decarboxylase expands its substrate scope towards the synthesis of the (R)-enantiomers of sterically hindered carboxylic acids]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722948v1?rss=1">
<title>
<![CDATA[
Ethanol-assisted core-shell microparticles for enzyme stabilization with precise size control 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722948v1?rss=1
</link>
<description><![CDATA[
In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions.
]]></description>
<dc:creator><![CDATA[ Yang, E., Khongkomolsakul, W., Dadmohammadi, Y., Abbaspourrad, A. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722948</dc:identifier>
<dc:title><![CDATA[Ethanol-assisted core-shell microparticles for enzyme stabilization with precise size control]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.723055v1?rss=1">
<title>
<![CDATA[
Schizokinen siderophores in the methylotrophy model organism Methylorubrum extorquens AM1 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.723055v1?rss=1
</link>
<description><![CDATA[
The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.
]]></description>
<dc:creator><![CDATA[ Sottorff, I. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.723055</dc:identifier>
<dc:title><![CDATA[Schizokinen siderophores in the methylotrophy model organism Methylorubrum extorquens AM1]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722510v1?rss=1">
<title>
<![CDATA[
Rhodopsin is a tunable capacitor buffering the toxic, desensitizing retinoids of the vertebrate eye 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722510v1?rss=1
</link>
<description><![CDATA[
Visual sensitivity creates photodamage risk, a trade-off thought to limit photo-resilience. Here we reveal that the locus of sensitivity, the visual pigment rhodopsin, moonlights as a tunable mechanism of photoprotection. Light activated rhodopsin (R*) mitigates phototoxicity and boosts rod sensitivity by serving as an overflow capacitor buffering all-trans retinal (atRAL), a toxic and desensitizing retinoid agonist that accumulates as lipofuscin, a clinical marker of macular degeneration. We show that R* stability does not guarantee increased signaling as previously proposed. Instead, across mammals R* stability reflects atRAL binding affinity (capacitance) tuned by photodamage risk. R* capacitance affords cytoprotection and, counterintuitively, promotes dark adaptation by shielding neighboring dark-state receptors from agonist interference. We treated a mouse model of defective atRAL clearance with a synthetic R* of unnaturally high atRAL capacitance. This gene therapy preserved retinal function following light damage and provided supra-physiological scotopic sensitivity despite being a signal-silent receptor, modulating endogenous R* signaling. During recent human evolution, rhodopsin mutations that enhance capacitance and cytoprotection have emerged in high irradiance environments and are now significantly associated with a 36% reduced risk of blindness. Together, our findings redefine rhodopsin as a tunable light buffer that can be leveraged to enhance photoreceptor function beyond natural limits.
]]></description>
<dc:creator><![CDATA[ Dbouk, N. H., Bagshaw, M., Bose, A. A., Rasdall, M., Arner, A., Zhao, K., Taylor, K. E., Praveen, N., Huo, W., Bautista, I., Musayeva, N., Xue, L., Hayes, J. N., McElhinney, M., Bick, A. G., Lea, A. J., Rex, T. S., Castiglione, G. M. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722510</dc:identifier>
<dc:title><![CDATA[Rhodopsin is a tunable capacitor buffering the toxic, desensitizing retinoids of the vertebrate eye]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722603v1?rss=1">
<title>
<![CDATA[
Thermal-Acoustic Activation of Hydrophobic Polystyrene Supports for High-Efficiency Aqueous Solid-Phase Peptide Synthesis 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722603v1?rss=1
</link>
<description><![CDATA[
Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However its reliance on hazardous organic solvents such as N N dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here we demonstrate that industrially relevant 2 chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate free 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) and Oxyma activation system with a synergistic thermal acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel forming peptides, opioid peptides derived from enkephalins, toxin inspired sequences, and a lipid interacting fragment of alpha synuclein. Analytical characterization by HPLC and MALDI TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.
]]></description>
<dc:creator><![CDATA[ Krishnan, S., Kambekar, A., Khandelwal, J., Pushpavanam, K. S. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722603</dc:identifier>
<dc:title><![CDATA[Thermal-Acoustic Activation of Hydrophobic Polystyrene Supports for High-Efficiency Aqueous Solid-Phase Peptide Synthesis]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722824v1?rss=1">
<title>
<![CDATA[
Multi-Substrate Specificity of Isoflavone hydroxylases (GmIFH) Drive Isoflavonoid Diversification in Soybean 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722824v1?rss=1
</link>
<description><![CDATA[
Isoflavone hydroxylases (IFHs, CYP81E) convert isoflavone aglycones into their respective hydroxylated intermediates, which direct legume isoflavones into specialized defense pathways. In soybean, their functions have been studied mostly in the context of the daidzein-derived glyceollin biosynthesis. Here we combine metabolomics-guided feature mining, phylogenetic analysis, heterologous enzymology, structural elucidation, and in planta metabolite validation to determine the functional landscape of the soybean IFH family. Analysis of a soybean isoflavonoid-enriched metabolomic dataset revealed unidentified hydroxyisoflavone features that co-accumulated with glyceollins, indicating branch chemistry that is not well-recognized. The systematic characterization of the repertoire of soybean CYP81E has demonstrated that 9 out of 11 GmIFHs are catalytically active and collectively span both 2'- and 3'- hydroxylation of the major soybean isoflavone aglycones. Among them, GmIFH9A showed broad substrate scope and regioselectivity, yielding canonical and previously unknown hydroxylated isoflavone products. NMR and LC-MS/MS were used to identify and validate the hydroxylated isoflavone products as 2'-hydroxyglycitein and 2'-hydroxyformononetin, whose presence was also confirmed in soybean roots, thus confirming two of the hidden soybean isoflavonoid network metabolites. Kinetic studies also indicated that, although the majority of GmIFHs prefer daidzein and genistein as substrates, a few isoforms are active towards methoxylated isoflavones as well, indicating functional divergence in this expanded family. Our findings collectively redefine soybean IFHs as a multi-functional enzyme module that expands the hydroxyisoflavone chemical space and reveals new biosynthetic entry points beyond canonical glyceollin pathway.
]]></description>
<dc:creator><![CDATA[ Khatri, P., McDowell, T., Marsolais, F., Renaud, J., Dhaubhadel, S. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722824</dc:identifier>
<dc:title><![CDATA[Multi-Substrate Specificity of Isoflavone hydroxylases (GmIFH) Drive Isoflavonoid Diversification in Soybean]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.723147v1?rss=1">
<title>
<![CDATA[
LonP1 chaperone-like activity is ATPase independent and is mediated by its N-domain 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.723147v1?rss=1
</link>
<description><![CDATA[
The mitochondrial Lon protease is essential for proteostasis through ATP-dependent proteolysis and suppression of protein aggregation through an unknown mechanism. Here we show in three independent aggregation systems that human Lon protease (LonP1) directly interacts with fibrillar aggregates to prevent further aggregation: LonP1 binds amyloid fibrils and inhibits their growth, independently of its protease and ATPase activities. This aggregation inhibition depends on hexamer stability, and even the N-domain hexamer of LonP1 lacking all catalytic domains inhibited aggregation, which localizes its fibril-binding interface. We propose that chaperone deficiencies in LonP1 mutants that are associated with genetic disease, are caused by reduced hexamer stability or increased turnover. Our results clarify the observed dual protease and chaperone function of LonP1 by localizing them to different domains and separating the catalytic activities, thereby facilitating targeting the specific functionalities. Further, we identify the structure of the chaperone substrate to be fibrillar aggregates, suggesting that LonP1 may protect against amyloid fibrils in healthy individuals.
]]></description>
<dc:creator><![CDATA[ Abrahams, J. P., Ahrensback Roesgaard, M., Armbruster, P., Sharpe, T., Schenck, N. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.723147</dc:identifier>
<dc:title><![CDATA[LonP1 chaperone-like activity is ATPase independent and is mediated by its N-domain]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723151v1?rss=1">
<title>
<![CDATA[
Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723151v1?rss=1
</link>
<description><![CDATA[
Pregnant women are frequently exposed to various endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), causing harm to both the developing placenta and fetus. BPA can promote placental dysfunction by altering key cellular processes such as differentiation, invasion, and migration in trophoblast cells. These cellular processes are also tightly managed by the ubiquitin proteasomal system via maintenance of the ubiquitinated protein pool. However, the BPA-mediated dysregulation of this ubiquitin proteasomal homeostasis is poorly understood. Therefore, we identified 19 deubiquitinases (DUBs) and a dynamic ubiquitinome profile of extravillous trophoblast cells (HTR8/SVneo), which reduced trophoblast cell migration post-BPA exposure. Further investigation using an integrated substrate-ligase-deubiquitinase network shows that BPA binding to PPAR-alpha or indirect regulation of its E3 Ligase MuRF1 and DUB USP5 via BPA resulted in hyper-ubiquitination of PPAR-alpha, triggering its nuclear localization. In the nucleus, the ubiquitinated PPAR-alpha can deregulate its migration-associated target gene expression, causing a reduction in the migration of HTR8/SVneo cells. This physiological alteration of extravillous trophoblast cells (EVTs) through BPA can disrupt placental homeostasis. Hence, we assumed that BPA-induced cellular alteration in EVTs can promote placental defects, which might contribute to adverse pregnancy outcomes.
]]></description>
<dc:creator><![CDATA[ Biswas, A., Saha, S., Sarmadhikari, D., Bisht, K. S., Asthana, S., Maiti, T. K. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723151</dc:identifier>
<dc:title><![CDATA[Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.722988v1?rss=1">
<title>
<![CDATA[
A CYFIP1-Inspired Peptidomimetic Modulates eIF4E-Dependent Translational Control in Cancer and Neurodevelopmental Disorders 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.722988v1?rss=1
</link>
<description><![CDATA[
The eukaryotic translation initiation factor 4E (eIF4E) is a central regulator of cap-dependent translation and a compelling pharmacological target in disorders marked by protein synthesis dysregulation, including cancer and Fragile X Syndrome (FXS). Among endogenous eIF4E regulators, the CYFIP1-eIF4E interaction is uniquely selective, offering a framework for designing targeted translation modulators. Here, we report Cy-9B, a rationally engineered, stapled peptidomimetic derived from CYFIP1 that binds eIF4E, disrupts the eIF4E-eIF4G complex, and suppresses cap-dependent translation. Enhanced-sampling free-energy simulations reveal that Cy-9B engages eIF4E through a non-canonical binding mode. Cy-9B exhibits drug-like properties, including high proteolytic stability and nanomolar affinity. Functionally, Cy-9B inhibits lung cancer cell proliferation, migration, and invasion. In neurodevelopmental disease models, Cy-9B partially normalizes excessive translation in FXS hippocampal neurons and rescues social behavior deficits in a Cyfip1 haploinsufficient Drosophila melanogaster model, restoring wildtype-like performance. Cy-9B emerges as a first-in-class therapeutic candidate for disorders sharing translational dysregulation, highlighting targeted modulation of eIF4E as a broadly applicable and physiologically compatible therapeutic strategy.
]]></description>
<dc:creator><![CDATA[ Romagnoli, A., Alsina, O., Raniolo, S., Gori, A., Foka, K., De Luca, A., Sgrignani, J., Rexha, J., Roscioni, A., Bergamaschi, G., Boussadia, Z., Pepponi, R., Venturini, G., Cavalli, A., Borsello, T., Martire, A., La Teana, A., Bagni, C., Di Marino, D., Limongelli, V. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.722988</dc:identifier>
<dc:title><![CDATA[A CYFIP1-Inspired Peptidomimetic Modulates eIF4E-Dependent Translational Control in Cancer and Neurodevelopmental Disorders]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.723405v1?rss=1">
<title>
<![CDATA[
Local aromatic interactions define temperature sensitivity of phase separation in an intrinsically disordered protein. 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.723405v1?rss=1
</link>
<description><![CDATA[
Liquid liquid phase separation (LLPS) of intrinsically disordered proteins is highly sensitive to environmental conditions, yet the molecular basis of sharp temperature responsiveness remains poorly understood. Here, we investigate a sequence encoded mechanism underlying the pronounced temperature sensitivity of phase separation using Sup35NM, the intrinsically disordered domain of the yeast prion Sup35. We show that a tyrosine rich local structural region within this domain encodes strong temperature responsiveness of droplet formation. Mutational analyses reveal that tyrosine residues mediate both intramolecular interactions that stabilize local structure and intermolecular interactions required for LLPS. Substitution of the tyrosine residues with alanine disrupts local structure and weakens intermolecular interactions, thereby diminishing temperature sensitivity. In contrast, substitution with phenylalanine promotes rapid droplet gelation, abolishes internal fluidity, suppresses amyloid formation, and confers resistance to temperature induced dissolution. Based on these findings, we propose a molecular model in which finely tuned, moderately weak aromatic interactions among tyrosines enable reversible local compaction that is sensitive to temperature, thereby generating a sharp phase transition. These results suggest that amino acid sequences encode not only phase separation propensity but also the sensitivity of condensates to environmental perturbations, providing a framework for understanding how intrinsically disordered proteins act as molecular sensors of cellular conditions.
]]></description>
<dc:creator><![CDATA[ Ohhashi, Y., Nishinami, S., Maruyama, Y., Fukuyama, M., Shiraki, K., Chatani, E., Taguchi, H. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.723405</dc:identifier>
<dc:title><![CDATA[Local aromatic interactions define temperature sensitivity of phase separation in an intrinsically disordered protein.]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.723350v1?rss=1">
<title>
<![CDATA[
Assessing State-Specific Accuracy of Cofolding Models for Kinases and GPCRs 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.723350v1?rss=1
</link>
<description><![CDATA[
AI-driven cofolding models have emerged as powerful tools for predicting protein-ligand complexes, yet whether ligand placement faithfully captures the conformational states of dynamic proteins remains unclear. Here we show that cofolding adaptively remodels binding pockets around bound ligands, but that this local accuracy is frequently decoupled from recovery of the broader conformational state. We benchmark four models, AlphaFold3, RosettaFold3, Boltz-2, and Chai-1, against a set of kinases and class A G protein-coupled receptors (GPCRs), protein families whose pharmacology depends on well-defined structural states. We find that even when ligand root-mean-square deviation (RMSD) is low, critical state markers, including kinase activation-loop geometries and GPCR intracellular arrangements, are frequently mispredicted. Incorporating state-annotated templates and filtered multiple sequence alignments (MSAs) improves conformational recovery in selected cases, yet weakly impacts others. Furthermore, while orthosteric ligand placement is generally reliable, allosteric binders expose a consistent blind spot across all models. These findings establish conformational decoupling as a fundamental limitation of current cofolding approaches, with direct implications for state-selective drug design
]]></description>
<dc:creator><![CDATA[ Obendorf, L., Doering, N. P., Knaus, P., Wolber, G. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.723350</dc:identifier>
<dc:title><![CDATA[Assessing State-Specific Accuracy of Cofolding Models for Kinases and GPCRs]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.723354v1?rss=1">
<title>
<![CDATA[
Systematic Characterization of Thermal Stability Assay Parameters and Application in Discovery of Peptide-Protein Interactions 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.723354v1?rss=1
</link>
<description><![CDATA[
Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of ~9,000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5-2 degrees Celsius proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to optimize PISA then apply the optimized conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation, and extends the utility of PISA beyond small molecules to dissecting peptide-protein interactions, an increasingly important modality in drug discovery.
]]></description>
<dc:creator><![CDATA[ Richards, D. M., zhai, F., Li, S., Yu, Q. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.723354</dc:identifier>
<dc:title><![CDATA[Systematic Characterization of Thermal Stability Assay Parameters and Application in Discovery of Peptide-Protein Interactions]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722977v1?rss=1">
<title>
<![CDATA[
Stable isotope-assisted computational mass spectrometry reveals root-specific alkaloids in Glycyrrhiza species 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722977v1?rss=1
</link>
<description><![CDATA[
Licorice (Glycyrrhiza) is a medicinal plant widely used in approximately 70% of traditional Japanese Kampo formulations and is known to produce a wide array of specialized metabolites with diverse pharmacological properties. Although hundreds of metabolites have been reported, the overall chemical diversity of Glycyrrhiza remains poorly characterized. Here, using mass spectrometry data obtained from fully 13C-labeled leaves and roots of Glycyrrhiza uralensis and Glycyrrhiza glabra, we determined the carbon number, followed by molecular formula and substructure prediction in combination with MS/MS similarity-based molecular networking. After excluding redundant ions, including isotopic peaks, adducts, and in-source fragments, we extracted 3,060 unique metabolite features with assigned carbon numbers. Among these, substructure information was assigned to 1,015 features (33%) across the four plant tissues, revealing the tissue-specific metabolome profiles. Furthermore, we discovered five previously unreported alkaloids, homopipecolic acid-conjugated flavonoids, in the roots of G. uralensis and G. glabra, and Glycine max, another member of the Fabaceae family. Two of these structures were validated using nuclear magnetic resonance spectroscopy. We further proposed a biosynthetic route involving a spontaneous reaction between 1-piperideine and malonyl glycoside substrates and confirmed the formation of the conjugated product using authentic standards.
]]></description>
<dc:creator><![CDATA[ Sawai, K., Todoroki, Y., Nakamukai, S., Matsuzawa, Y., Noguchi, K., Kato, T., Mori, T., Rai, A., Hirai, M. Y., Tsugawa, H. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722977</dc:identifier>
<dc:title><![CDATA[Stable isotope-assisted computational mass spectrometry reveals root-specific alkaloids in Glycyrrhiza species]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.723046v1?rss=1">
<title>
<![CDATA[
Identification of the siderophore schizokinen and its derivatives by LCHRMS and mass-tandem fragmentation 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.723046v1?rss=1
</link>
<description><![CDATA[
Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.
]]></description>
<dc:creator><![CDATA[ Sottorff, I. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.723046</dc:identifier>
<dc:title><![CDATA[Identification of the siderophore schizokinen and its derivatives by LCHRMS and mass-tandem fragmentation]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.07.721674v1?rss=1">
<title>
<![CDATA[
The turn less taken: Investigating patterns in β-turn dynamics using large-scale molecular dynamics data 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.07.721674v1?rss=1
</link>
<description><![CDATA[
{beta}-turns are among the most common structural motifs in proteins, yet their conformational dynamics and sequence determinants remain incompletely understood. Here we present a data-driven classification and dynamic analysis of {beta}-turn conformations using large-scale molecular dynamics trajectories from the mdCATH database. Clustering of backbone dihedral angles using a cross-bond Ramachandran representation identifies six {beta}-turn types, including a previously uncharacterized hybrid I/I' cluster that combines geometric features of canonical type I and I' conformations. Time-resolved analysis indicates that this hybrid state acts as a transient intermediate state of {beta}-turns. Transitions observed in molecular dynamics simulations closely match NMR ensembles and altlocs detected in X-ray crystal structures, with the most dominant exchanges occurring between type I and II, and between type I' and II' turns. Sequence analysis shows that each turn type exhibits characteristic amino acid preferences at the central residues (i+1 and i+2). Within these overall preferences, specific residue pairs display distinct biases toward static or dynamic behavior. Targeted in silico substitutions that interchange dynamic- and static-enriched residue pairs shift the conformational behavior of turns accordingly, providing direct support for these sequence-dynamics relationships. Analysis of flanking secondary-structure environments reveals that structural context further modulates turn flexibility, with strand- and coil-associated turns exhibiting higher dynamic propensity than helix-associated turns. Together, these results reveal how sequence composition and structural context jointly shape the conformational landscape of {beta}-turns.
]]></description>
<dc:creator><![CDATA[ Zhang, S., Maddipatla, S. A., Vedula, S., Marx, A., Bronstein, A. M. ]]></dc:creator>
<dc:date>2026-05-08</dc:date>
<dc:identifier>doi:10.64898/2026.05.07.721674</dc:identifier>
<dc:title><![CDATA[The turn less taken: Investigating patterns in β-turn dynamics using large-scale molecular dynamics data]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.723016v1?rss=1">
<title>
<![CDATA[
Evolution of Protein Regulation in the Vertebrate Glucose Sensor 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.723016v1?rss=1
</link>
<description><![CDATA[
Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the body's glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzyme's conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a de novo loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions.
]]></description>
<dc:creator><![CDATA[ Kamalaldinezabadi, S. S., Santiago, J. I., Papa, J. E., Wang, Y., Frantom, P. A., Li, H., Silvers, R., Whittington, A. C., Miller, B. G. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.723016</dc:identifier>
<dc:title><![CDATA[Evolution of Protein Regulation in the Vertebrate Glucose Sensor]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.04.721528v1?rss=1">
<title>
<![CDATA[
Glycosylation-dependent Turnover of Triterpenoid Saponins Controls Insect Deterrence 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.04.721528v1?rss=1
</link>
<description><![CDATA[
Background and Aims: Plants deploy triterpenoid saponins as chemical defences against herbivores, yet it remains unclear whether insect digestion detoxifies these compounds or generates equally or more active metabolites. Because saponin bioactivity depends strongly on glycosylation patterns, we examined the fate and defensive activity of hederagenin-derived saponins during herbivory. Methods: Larvae of Plutella xylostella were fed leaf discs containing structurally defined hederagenin-derived saponins. Saponin composition in treated leaves and larval frass was analysed by LC-qTOF-ESI-MS/MS. Feeding assays were used to compare the antifeedant activity of mono- and bidesmosidic forms. Key Results: Larvae selectively metabolized complex hederagenin-derived saponins into simpler forms, with cellobiosides converted into monoglucosides during digestion, resulting in a marked shift in saponin composition between ingested material and frass. Feeding assays showed that monodesmosidic saponins strongly deterrer feeding, whereas bidesmosidic saponins were largely inactive. The loss of activity in bidesmosidic saponins was not explained by differential metabolism, indicating that glycosylation patterns directly determine biological function. Conclusions: Insect herbivores selectively modify saponin structures through deglycosylation, thereby altering their defensive properties. Our findings demonstrate that glycosylation governs both saponin activity and metabolic fate, highlighting insect-driven turnover as a critical component of plant chemical defence during plant-herbivore interactions. Keywords: triterpenoid saponins, hederagenin, glycosylation, deglycosylation, Barbarea vulgaris, Plutella xylostella, antifeedant activity, chemical ecology
]]></description>
<dc:creator><![CDATA[ Shen, J., Cardenas, P. D., Bak, S. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.04.721528</dc:identifier>
<dc:title><![CDATA[Glycosylation-dependent Turnover of Triterpenoid Saponins Controls Insect Deterrence]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.04.722805v1?rss=1">
<title>
<![CDATA[
A method to synthesize analytical rhodoquinone standards for quantitative analysis in tissue specimen 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.04.722805v1?rss=1
</link>
<description><![CDATA[
Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.
]]></description>
<dc:creator><![CDATA[ Do, T., Ali, A., Spinelli, J. B. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.04.722805</dc:identifier>
<dc:title><![CDATA[A method to synthesize analytical rhodoquinone standards for quantitative analysis in tissue specimen]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.06.722604v1?rss=1">
<title>
<![CDATA[
DNA supercoiling modulates bZIP transcription factor/DNA interaction 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.06.722604v1?rss=1
</link>
<description><![CDATA[
DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including BioLayer Interferometry applied here for the first time to topology-dependent protein/DNA interactions, we show that DNA supercoiling directly reshapes GCN4/DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein/DNA interactions.
]]></description>
<dc:creator><![CDATA[ Valenti, A., Mattossovich, R., Merlo, R., Schifino, G., Sandomenico, A., Widersten, M., Calio, A., Peters, J., Pastore, A., Parracino, A. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.06.722604</dc:identifier>
<dc:title><![CDATA[DNA supercoiling modulates bZIP transcription factor/DNA interaction]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.04.720848v1?rss=1">
<title>
<![CDATA[
Leiomodin 2 functions as a processive pointed-end elongator of actin filaments 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.04.720848v1?rss=1
</link>
<description><![CDATA[
The actin cytoskeleton drives essential processes like cell migration and muscle contraction. While barbed-end polymerization is well-established, pointed-end elongation was long considered impossible in vivo. Here, we demonstrate that Leiomodin 2 (Lmod2), which localizes to thin-filament pointed ends (PEs) in striated muscle cells, functions as the first identified eukaryotic processive actin polymerase. Single-molecule and single-filament imaging reveal that Lmod2 stably associates with PEs in vitro, enabling elongation even in the presence of high profilin concentrations found in the cytoplasm that otherwise would cause depolymerization of free PEs. We find that both processivity and elongation rate of Lmod are dependent on its WH2 domain. Remarkably, human dilated cardiomyopathy-associated mutations in Lmod2 greatly reduce Lmod2's PE elongation activity, providing a potential mechanism for disease progression, underscoring the essential role of its actin polymerase activity in formation and maintenance of muscle sarcomeres.
]]></description>
<dc:creator><![CDATA[ Biswas, S., Larrinaga, T. M., Choubey, S., Gregorio, C. C., Shekhar, S. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.04.720848</dc:identifier>
<dc:title><![CDATA[Leiomodin 2 functions as a processive pointed-end elongator of actin filaments]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.04.722819v1?rss=1">
<title>
<![CDATA[
A lipid-binding protein in black-legged tick saliva selectively recognizes Borrelia burgdorferi lipids 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.04.722819v1?rss=1
</link>
<description><![CDATA[
A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.
]]></description>
<dc:creator><![CDATA[ Shi, W. O., MacMackin-Ingle, T., Perez, M. W., Griffith, W. P., Chen, L., Seshu, J., Renthal, R. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.04.722819</dc:identifier>
<dc:title><![CDATA[A lipid-binding protein in black-legged tick saliva selectively recognizes Borrelia burgdorferi lipids]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.05.05.722991v1?rss=1">
<title>
<![CDATA[
Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.05.05.722991v1?rss=1
</link>
<description><![CDATA[
CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Pseudoalteromonas (PseCAST) shows uniquely robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the PseCAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of PseTnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.
]]></description>
<dc:creator><![CDATA[ Walter, M., Finocchio, G., Oberli, S., Hammerschmid, I. C., Lampe, G. D., Karan, J., Swartjes, T., Sternberg, S. H., Jinek, M., Querques, I. ]]></dc:creator>
<dc:date>2026-05-07</dc:date>
<dc:identifier>doi:10.64898/2026.05.05.722991</dc:identifier>
<dc:title><![CDATA[Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-05-07</prism:publicationDate>
<prism:section></prism:section>
</item>
</rdf:RDF>
