Biology subjects

Zhu, M.

Publications and source records attributed to Zhu, M..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

The pan-genome and local adaptation of Arabidopsis thaliana

Arabidopsis thaliana has been used as a model species for research in a diverse collection of plant species. However, previous studies based on single reference genomes and short-read sequencing data are restricted to detecting variable genes and large structural variation (SV) underlying local adaptation. Here we de novo assemble high-quality chromosomal genomes of 38 A. thaliana ecotypes (with 6 relict ones) using PacBio-HiFi long-read sequencing. From these newly assembled genomes, we annotate several thousand new genes through pan-genomic analysis in comparison to the previous reference genome. The identified variable genes are mainly enriched in and associated with ecological adaptation and this species substantially expands its gene repertoire for local adaptation. We construct a graph-based pan-genome and identify 62,525 SVs which overlap with 14,243 genes. These genes are enriched in multiple ecological adaptation functions, including secondary metabolic processes, enzyme regulation, and biotic/abiotic stimulus. For example, a 566 bp insertion in the promoter of the light-adaptation KNAT3 gene was specific to the high-altitude relict Tibet-0 ecotype. This SV reduces the expression level of KNAT3 and promotes A. thaliana adaptation to habitats high in light radiation. In addition, compared with the SNPs, the SVs identified in this study captured the missing heritability and we detected novel SV associations with environmental variables in their native range, highlighting the value of SVs in environmental adaptation. The genome resources presented here will help pinpoint genetic changes that include both SVs and the ecotype-specific genes for local adaptation of A. thaliana and increase our understanding of the molecular mechanisms in this model species to respond to varied habitats.

genomics↗

bioRxiv2022

Shift of the insoluble content of the proteome in aging mouse brain

Aging and protein aggregation diseases are inextricably linked. During aging, cellular response to unfolded proteins are believed to decline which results in diminished protein homeostasis (proteostasis). Indeed, in model organisms, such as C. elegans, proteostatic decline with age has even been linked to the onset of aggregation of proteins in wild-type animals. However, this correlation has not been extensively characterized in aging mammals. To reveal the insoluble portion of the proteome, we analyzed the detergent-insoluble fraction of mouse brain tissues after high-speed centrifugation by quantitative mass spectrometry. We identified a cohort of 171 proteins enriched in the pellet fraction of older mice including the alpha crystallin small heat shock protein. We then performed a meta-analysis to compare features among distinct groups of detergent-insoluble proteins reported in the literature. Surprisingly, our analysis revealed that features associated with proteins found in the pellet fraction differ depending on the ages of the mice. In general, insoluble proteins from young models (<15 weeks) were more likely to be RNA-binding, more disordered and more likely to be found in membraneless organelles. These traits become less prominent with age within the combined dataset, as proteins with more structure enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a specific group of proteins to enter the pellet fraction as a result of loss of proteostasis.

cell biology↗

bioRxiv2022

Resilient CD8+ T cells maintain a high cytotoxic capacity by balancing ROS via ME1 upregulation

Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignances. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysisindependent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/517988v3_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@12e0f36org.highwire.dtl.DTLVardef@e8d412org.highwire.dtl.DTLVardef@1baf57eorg.highwire.dtl.DTLVardef@14b2b43_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICX3CR1+ and low Dy m identify functional resilient CD8+ T cells. C_LIO_LIResilient CD8+ T cells are highly cytotoxic and have less ROS. C_LIO_LIResilient CD8+ T cells express more ME1 that can balance extra ROS. C_LIO_LIME1 overexpression can promote CTL function of CD8+ T cells. C_LI

immunology↗

bioRxiv2022

Integrated Network Pharmacology and Experimental Analysis Unveil Modulation of EGFR/MAPK Signaling Cascades in Acute Cerebral Ischemia-Reperfusion Injury by Qing-Tong-Hua-Yu Decoction

ObjectiveBased on network pharmacology, the response of Qing-tong-hua-yu Decoction (QTHY) to the regulation of EGFR/MAPK signaling cascade in cerebral ischemia-reperfusion injury was discussed and the possible mechanism of the protective effect of QTHY on the cerebral ischemia-reperfusion injury was studied. MethodsA compound-target disease-function-pathway network was established and analyzed based on the network pharmacology approach used in Chinese medicine. The correlation, which is between effect of the components of QTHY Decoction against CI/RI with EGFR/MAPK signalling cascade response, was observed. And then the degree of neurological deficits in each group was assessed after cerebral ischemia for 2 hours and reperfusion for 3 hours, 24 hours, 3 days and 7 days. Expression levels of EGFR and p44/42MAPK in ischemic brain tissue at different time points in various groups of rats were tested by Western bolt (WB), real-time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC). ResultsNetwork pharmacology analysis revealed that QTHY-mediated treatment involved 439 key targets, in which the effect of QTHY groups against CI/RI was associated with EGFR/MAPK signaling cascade. QTHY treatment reduced neurological deficit scores and improved ischemic changes in rats. In addition, QTHY promoted EGFR and p44/42MAPK expression in the SVZ through the EGFR/MAPK signaling cascade, with varying degrees of improvement at different time points. ConclusionQTHY can better improve cerebral ischemia injury in CI / RI rats and exert the neuroprotective effect of cerebral ischemia-reperfusion injury. This may be related to the potential of QTHY to activate the EGFR / MAPK signaling cascade, which is consistent with the results of network pharmacology analysis.

neuroscience↗

bioRxiv2022

Patterning of phase-separated condensates by Dnd1 controls cell fate

Germ granules, condensates of phase-separated RNA and protein, are essential for germline development, but how these molecules are organized within the granules and whether such an organization is relevant for germ cell fate is unclear. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the vertebrate-specific Dead end protein is essential for positioning nanos3 RNA at the condensates periphery, where ribosomes are located. Without Dead end, or when translation is inhibited, nanos3 RNA translocates into granule interiors, far from the ribosomes location. These findings reveal the molecular mechanisms controlling the spatial organization of RNA within the phase-separated organelle and the importance of subgranule RNA localization for preserving germ cell totipotency.

cell biology↗

bioRxiv2022

IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration

While midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated a Igfbp2-CreER knockin strain, which specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21% to 41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with DDC, IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration. HighlightsO_LILineage tracing showed that midlobular hepatocytes proliferate during homeostasis. C_LIO_LIZone 2 cells were protected from portal and centrilobular injuries and replaced lost hepatocytes. C_LIO_LIFasting induced significant changes in liver zonation. C_LI eTOC statementThe liver consists of different zones with spatial heterogeneity in their metabolic functions. Here, a new Igfbp2-CreER line enabled direct tracing of midlobular hepatocytes, which showed that IGFBP2+ cells serve as a source of new hepatocytes during normal homeostasis and regeneration.

developmental biology↗

bioRxiv2022

Sex differences in the impact of electronic nicotine vapor on corticotropin-releasing factor receptor 1 neurons in the mouse ventral tegmental area

Nicotine engages dopamine neurons in the ventral tegmental area (VTA) to encode reward and drive the development of nicotine addiction, however how nicotine selectively alters other VTA populations remains to be determined. Here, we used male and female CRF1-GFP mice and nicotine vapor exposure to examine the effects of nicotine in VTA corticotropin-releasing factor receptor 1 (CRF1) neurons. We use immunohistochemistry and electrophysiology to examine neuronal activity, excitability, and inhibitory signaling. We found that VTA CRF1 neurons are mainly dopaminergic and project to the nucleus accumbens (VTA-NAcCRF1 neurons). VTA-NAcCRF1 neurons show greater phasic inhibition in naive females and greater focal nicotine-induced increases in firing in naive males. Following acute nicotine vapor exposure, phasic inhibition was not altered, but focal nicotine-induced tonic inhibition was enhanced in females and diminished in males. Acute nicotine vapor exposure did not affect firing in VTA-NAcCRF1 neurons, but females showed lower baseline firing and higher focal nicotine-induced firing. Activity (cFos) was increased in the CRF1 dopaminergic VTA population in both sexes, but with greater increases in females. Following chronic nicotine vapor exposure, both sexes displayed reduced basal phasic inhibition and the sex difference in tonic inhibition following acute vapor exposure was no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated in either sex. These findings reveal sex- and exposure-dependent changes in mesolimbic VTA-NAc CRF1 neuronal activity, inhibitory signaling, and nicotine sensitivity following nicotine vapor exposure. These changes potentially contribute to nicotine-dependent behaviors and the intersection between stress, anxiety, and addiction.

neuroscience↗

bioRxiv2022

The small cell lung cancer neuroendocrine transdifferentiation explorer

SCLC is a high-grade neuroendocrine (NE) cancer that exhibits cellular plasticity. Transdifferention into non-NE cells creates considerable intra-tumoral heterogeneity, enhanced metastasis, greater tumor burden, and treatment resistance. Similar NE transdifferentiation has been observed in neuroblastoma (NBL). Targeting NE plasticity and cooperation between NE and non-NE cells in the tumor microenvironment may provide an avenue to enhance and restore sensitivity to available treatments. Although substantial transcriptomic changes take place upon NE transdifferentiation, conservation of these changes has not been investigated. In this study, we extensively curated genes associated with NE transdifferentiation in SCLC. We collected 35 datasets and compared the NE score-associated transcriptome across studies, for SCLC vs. NBL human tumors, human NBL tumors vs. cell lines, SCLC human tumors vs. tumors from genetically engineered mouse models (GEMMs), and SCLC GEMM uncultured cancer cells vs. cultured cancer cells. We have also created a user-friendly web application for researchers to explore these results. This work establishes a useful resource for researchers to understand the NE transdifferentiation landscape and explore context-dependent NE associations in SCLC and NBL.

cancer biology↗

bioRxiv2022

Epinephrine inhibits PI3K alpha via the Hippo kinases.

The phosphoinositide 3-kinase, p110, is an essential mediator of insulin signaling and glucose homeostasis. We systematically interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110 and found that the Hippo kinases phosphorylate and completely inhibit its activity. This inhibitory state corresponds to a conformational change of a membrane binding domain on p110, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases, MST1/2, using forskolin or epinephrine is associated with phosphorylation and inhibition of p110, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules. Our study reveals a novel inhibitory pathway of PI3K signaling and a previously unappreciated link between epinephrine and insulin signaling.

physiology↗

bioRxiv2022

Dose-dependent suppression of hippocampal contextual memory formation, place cells, and spatial engrams by the NMDAR antagonist (R)-CPP

A common way to study the functional importance of N-methyl-D-aspartate receptors (NMDARs) in hippocampal memory-encoding circuits is by administering NMDAR antagonists. We recently compared the effects of (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), a competitive NMDAR antagonist, on suppression of memory in vivo versus suppression of NMDAR-mediated field EPSPs (fEPSPNMDA) and long-term potentiation (LTP) in vitro. Surprisingly, we found that concentrations that block contextual conditioning in vivo are ineffective at blocking the fEPSPNMDA or LTP in vitro. Here we tested one possible explanation for the mismatch - that the hippocampus is relatively resistant to CPP compared to other brain structures engaged in contextual fear conditioning. We used the context pre-exposure facilitation effect (CPFE) paradigm to isolate the hippocampal component of contextual learning, and in-vivo calcium imaging of place cells and spatial engrams to directly assess hippocampal spatial coding. We found that, by both measures, the active enantiomer (R)-CPP did interfere with hippocampal function at concentrations below those that block fEPSPs or LTP. We conclude that the alternative - that CPP interferes with memory by targeting NMDARs in interneurons rather than pyramidal neurons - is the more likely explanation.

neuroscience↗

bioRxiv2022

KDM4A promotes NEPC progression through regulation of MYC expression

Neuroendocrine prostate cancer (NEPC) represents one of the most lethal forms of prostate cancer (PCa) and lacks life-prolonging treatment. The incidence of NEPC is increased due to the widespread use of AR pathway inhibitors (ARPIs) in the treatment of non-metastatic CRPC and hormone-sensitive metastatic tumors. Here, we identified histone lysine demethylase KDM4A as a key player in NEPC progression and an effective therapeutic target. We found that KDM4A mRNA and protein are overexpressed in human and mouse NEPC compared to prostate adenocarcinoma. Knockdown (KD) or knockout (KO) of KDM4A in NEPC cell lines suppressed cancer cell growth in vitro and in vivo. Mechanistically, we found that KDM4A promotes NEPC progression, in part, through direct transcriptional regulation of MYC. We showed that MYC is hyper-activated in human and mouse NEPC. KDM4A KD led to suppression of MYC signaling. MYC KD or inhibition profoundly suppressed NEPC cell proliferation. Furthermore, a potent pan-KDM4 inhibitor QC6352 significantly reduced NEPC cell growth in vitro and in vivo. Taken together, we demonstrated that KDM4A is an important regulator of NEPC progression and targeting KDM4A may potentially be an effective therapeutic strategy for NEPC. SignificanceNeuroendocrine prostate cancer (NEPC) is a highly aggressive prostate cancer subtype that is resistant to potent androgen receptor pathway inhibitors (ARPIs) and currently lacks effective therapeutic options. Histone lysine demethylase KDM4A is an important epigenetic regulator of gene expression in development and cancer. In this study, we show that KDM4A is highly expressed in NEPC and is required for NEPC proliferation, anchorage-independent growth, and in vivo growth, which is in part mediated through the regulation of MYC expression. Importantly, we demonstrate that inhibition of KDM4A significantly impairs NEPC growth in preclinical models. Thus, our findings provide valuable insights into the molecular mechanisms underlying NEPC progression and offer a rationale for clinical trials with KDM4 inhibitor in NEPC patients.

cancer biology↗

bioRxiv2022

In situ architecture and membrane fusion of SARS-CoV-2 Delta variant

Among the current five Variants of Concern, infections caused by the SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, architecture of intact Delta virions remains veiled. Moreover, molecular evidences for the detailed mechanism of S-mediated membrane fusion are missing. Here we reported the in situ structure and distribution of S on the authentic Delta variant, and discovered invagination in the distinctive Delta architecture. We also captured fusion snapshots from the virus-virus fusion events, provided structural evidences for Deltas attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S over that of the Wuhan-Hu-1 S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant. In BriefCryo-ET of intact SARS-CoV-2 Delta variant revealed its distinctive architecture and captured snapshots of its membrane fusion in action.

biophysics↗

bioRxiv2022

NVX-CoV2373 vaccination induces functional SARS-CoV-2-specific CD4+ and CD8+ T cell responses

NVX-CoV2373 is an adjuvanted recombinant full-length SARS-CoV-2 spike trimer protein vaccine demonstrated to be protective against COVID-19 in efficacy trials. Here we demonstrate that vaccinated subjects made CD4+ T cell responses after one and two doses of NVX-CoV2373, and a subset of individuals made CD8+ T cell responses. Characterization of the vaccine-elicited CD8+T cells demonstrated IFN{gamma} production. Characterization of the vaccine-elicited CD4+ T cells revealed both circulating T follicular helper cells (cTFH) and TH1 cells (IFN{gamma}, TNF, and IL-2) were detectable within 7 days of the primary immunization. Spike-specific CD4+ T cells were correlated with the magnitude of the later SARS-CoV-2 neutralizing antibody titers, indicating that robust generation of CD4+ T cells, capable of supporting humoral immune responses, may be a key characteristic of NVX-CoV2373 which utilizes Matrix-M adjuvant.

immunology↗

bioRxiv2022

LTD-inducing low frequency stimulation enhances p-Tau181 and p-Tau217 in an age-dependent manner in live rats

The progressive cognitive decline in Alzheimers disease (AD) patients correlates with the extent of tau pathology, in particular tau hyperphosphorylation, which is strongly age-associated. Although elevation of phosphorylated tau (p-Tau) on residues Thr181 (p-Tau181), Thr217 (p-Tau217), and Thr231 (p-Tau231) in cerebrospinal fluid or blood are recently proposed to be particularly sensitive markers of early AD, the generation of p-Tau during brain activity is poorly understood. A major form of synaptic plasticity, long-term depression (LTD), has recently been linked to the enhancement of tau phosphorylation. Here we show that low frequency stimulation (LFS), used to induce LTD, enhances p-Tau181 and p-Tau217 in an age-dependent manner in the hippocampus of live rats. In contrast, phosphorylation at residues Thr231, Ser202/Thr205, and Ser396 is less sensitive to LFS. Pharmacological antagonism of either NMDA or metabotropic glutamate 5 (mGluR5) receptors inhibits the elevation of both p-Tau181 and p-Tau217. Targeting ageing with a small molecule cognitive enhancer ISRIB (trans-isomer) prevents the enhancement of p-Tau by LFS in aged rats. Together, our data provide an in vivo means to uncover brain plasticity-related cellular and molecular processes of tau phosphorylation in health and ageing conditions.

neuroscience↗

bioRxiv2022

Frequency-specific neural signatures of perceptual content and perceptual stability

In the natural environment, we often form stable perceptual experiences from ambiguous and fleeting sensory inputs. Which neural activity underlies the content of perception and which neural activity supports perceptual stability remains an open question. We used a bistable perception paradigm involving ambiguous images to behaviorally dissociate perceptual content from perceptual stability, and magnetoencephalography (MEG) to measure whole-brain neural dynamics in humans. Combining multivariate decoding and neural state-space analyses, we found frequency band-specific neural signatures that underlie the content of perception and promote perceptual stability, respectively. Across different types of images, non-oscillatory neural activity in the slow cortical potential (SCP, <5 Hz) range supported the content of perception. By contrast, perceptual stability is influenced by the amplitude of alpha and beta oscillations. In addition, neural activity underlying perceptual memory, which supports perceptual stability when sensory input is temporally removed from view, also encodes elapsed time. Together, these results reveal distinct neural mechanisms that support the content vs. stability of visual perception.

neuroscience↗

bioRxiv2022

A Cambrian soft-bodied conical animal illustrates the origins of lophophorate phyla

The origin and early evolution of lophotrochozoans remain a difficult but crucial issue in reconstructing metazoan phylogeny. Exceptionally preserved fossils have provided hopeful information for resolving this lophotrochozoan problem. Here we identify that Conicula striata, a soft-bodied conical animal from the early Cambrian Chengjiang Lagerstatte, has mosaic characteristics of different lophophorate phyla. C. striata possesses a phoronid-like vermiform trunk housing a U-shaped gut and a coiled lophophore comprising [≥]12 arms with numerous tentacles, but it also bears brachiopod-like features including an undivided mantle enclosing lophophore and a pedicle-like protrusion on annulated trunk. Phylogenetic analysis retrieves C. striata as a total-group lophophorate and as an intermediate taxon between Phoronida and Brachiopoda. This suggests that the bivalved architecture of brachiopods originated from an undivided mantle in a phoronid-like ancestor, and that the ancestral vermiform trunk became reduced during the origin of Brachiopoda, illuminating the origins of body plans in the lophophorate phyla.

paleontology↗

bioRxiv2022

Control of contextual memory through interneuronal α5-GABAA receptors

{gamma}-aminobutyric acid type A receptors that incorporate 5 subunits (5-GABAARs) are highly enriched in the hippocampus and are strongly implicated in control of learning and memory. Receptors located on pyramidal neuron dendrites have long been considered responsible, but here we report that their selective knockout from either interneurons (5-i-KO) or pyramidal neurons (5-pyr-KO) interferes with the ability of the general anesthetic etomidate to suppress contextual conditioning. Using Ca2+ imaging of CA1 pyramidal neuron activity in freely exploring mice to assess hippocampal function directly, we found that etomidate blocked the development of place cells and spatial engrams in wild type (WT) and 5-pyr-KO mice, but not in 5-i-KO mice. In addition, 5-i-KO mice developed weaker spatial engrams than WT mice under control conditions. These findings show that interneuronal 5-GABAARs serve a physiological role in promoting spatial learning, and that they mediate the suppression of hippocampus-dependent memory by etomidate.

neuroscience↗

bioRxiv2022

Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily

Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.

biochemistry↗