%0 Journal Article %J J Neurosci %D 2021 %T APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis. %A Ramakrishna, Sarayu %A Jhaveri, Vishwaja %A Konings, Sabine C %A Nawalpuri, Bharti %A Chakraborty, Sumita %A Holst, Bjørn %A Schmid, Benjamin %A Gouras, Gunnar K %A Freude, Kristine K %A Muddashetty, Ravi S %X

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis. Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

%B J Neurosci %V 41 %P 8686-8709 %8 2021 Oct 20 %G eng %N 42 %R 10.1523/JNEUROSCI.0435-21.2021 %0 Journal Article %J Stem Cell Reports %D 2021 %T Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3. %A Chandrasekaran, Abinaya %A Dittlau, Katarina Stoklund %A Corsi, Giulia I %A Haukedal, Henriette %A Doncheva, Nadezhda T %A Ramakrishna, Sarayu %A Ambardar, Sheetal %A Salcedo, Claudia %A Schmidt, Sissel I %A Zhang, Yu %A Cirera, Susanna %A Pihl, Maria %A Schmid, Benjamin %A Nielsen, Troels Tolstrup %A Nielsen, Jørgen E %A Kolko, Miriam %A Kobolák, Julianna %A Dinnyés, András %A Hyttel, Poul %A Palakodeti, Dasaradhi %A Gorodkin, Jan %A Muddashetty, Ravi S %A Meyer, Morten %A Aldana, Blanca I %A Freude, Kristine K %X

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

%B Stem Cell Reports %V 16 %P 2736-2751 %8 2021 Nov 09 %G eng %N 11 %R 10.1016/j.stemcr.2021.09.013 %0 Journal Article %J Cell Rep %D 2021 %T Correction of amygdalar dysfunction in a rat model of fragile X syndrome. %A Fernandes, Giselle %A Mishra, Pradeep K %A Nawaz, Mohammad Sarfaraz %A Donlin-Asp, Paul G %A Rahman, Mohammed Mostafizur %A Hazra, Anupam %A Kedia, Sonal %A Kayenaat, Aiman %A Songara, Dheeraj %A Wyllie, David J A %A Schuman, Erin M %A Kind, Peter C %A Chattarji, Sumantra %X

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in vitro and in vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

%B Cell Rep %V 37 %P 109805 %8 2021 Oct 12 %G eng %N 2 %R 10.1016/j.celrep.2021.109805 %0 Journal Article %J Science %D 2021 %T Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India. %A Dhar, Mahesh S %A Marwal, Robin %A Vs, Radhakrishnan %A Ponnusamy, Kalaiarasan %A Jolly, Bani %A Bhoyar, Rahul C %A Sardana, Viren %A Naushin, Salwa %A Rophina, Mercy %A Mellan, Thomas A %A Mishra, Swapnil %A Whittaker, Charles %A Fatihi, Saman %A Datta, Meena %A Singh, Priyanka %A Sharma, Uma %A Ujjainiya, Rajat %A Bhatheja, Nitin %A Divakar, Mohit Kumar %A Singh, Manoj K %A Imran, Mohamed %A Senthivel, Vigneshwar %A Maurya, Ranjeet %A Jha, Neha %A Mehta, Priyanka %A A, Vivekanand %A Sharma, Pooja %A Vr, Arvinden %A Chaudhary, Urmila %A Soni, Namita %A Thukral, Lipi %A Flaxman, Seth %A Bhatt, Samir %A Pandey, Rajesh %A Dash, Debasis %A Faruq, Mohammed %A Lall, Hemlata %A Gogia, Hema %A Madan, Preeti %A Kulkarni, Sanket %A Chauhan, Himanshu %A Sengupta, Shantanu %A Kabra, Sandhya %A Gupta, Ravindra K %A Singh, Sujeet K %A Agrawal, Anurag %A Rakshit, Partha %A Nandicoori, Vinay %A Tallapaka, Karthik Bharadwaj %A Sowpati, Divya Tej %A Thangaraj, K %A Bashyam, Murali Dharan %A Dalal, Ashwin %A Sivasubbu, Sridhar %A Scaria, Vinod %A Parida, Ajay %A Raghav, Sunil K %A Prasad, Punit %A Sarin, Apurva %A Mayor, Satyajit %A Ramakrishnan, Uma %A Palakodeti, Dasaradhi %A Seshasayee, Aswin Sai Narain %A Bhat, Manoj %A Shouche, Yogesh %A Pillai, Ajay %A Dikid, Tanzin %A Das, Saumitra %A Maitra, Arindam %A Chinnaswamy, Sreedhar %A Biswas, Nidhan Kumar %A Desai, Anita Sudhir %A Pattabiraman, Chitra %A Manjunatha, M V %A Mani, Reeta S %A Arunachal Udupi, Gautam %A Abraham, Priya %A Atul, Potdar Varsha %A Cherian, Sarah S %X

Delhi, the national capital of India, has experienced multiple SARS-CoV-2 outbreaks in 2020 and reached population seropositivity of over 50% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant B.1.617.2 (Delta) replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and reduced sensitivity to immune responses generated against earlier variants (median estimates; ×1.5-fold, 20% reduction). Seropositivity of an employee and family cohort increased from 42% to 87.5% between March and July 2021, with 27% reinfections, as judged by increased antibody concentration after a previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi.

%B Science %P eabj9932 %8 2021 Oct 14 %G eng %R 10.1126/science.abj9932 %0 Journal Article %J Mol Cell %D 2021 %T Proteome plasticity in response to persistent environmental change. %A Domnauer, Matthew %A Zheng, Fan %A Li, Liying %A Zhang, Yanxiao %A Chang, Catherine E %A Unruh, Jay R %A Conkright-Fincham, Juliana %A McCroskey, Scott %A Florens, Laurence %A Zhang, Ying %A Seidel, Christopher %A Fong, Benjamin %A Schilling, Birgit %A Sharma, Rishi %A Ramanathan, Arvind %A Si, Kausik %A Zhou, Chuankai %K Acclimatization %K Adaptation, Physiological %K Animals %K Environmental Exposure %K Gene Expression Regulation, Fungal %K Hot Temperature %K Proteome %K Saccharomycetales %K Stress, Physiological %K Transcriptome %X

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

%B Mol Cell %V 81 %P 3294-3309.e12 %8 2021 08 19 %G eng %N 16 %R 10.1016/j.molcel.2021.06.028 %0 Journal Article %J Nature %D 2021 %T SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. %A Mlcochova, Petra %A Kemp, Steven A %A Dhar, Mahesh Shanker %A Papa, Guido %A Meng, Bo %A Ferreira, Isabella A T M %A Datir, Rawlings %A Collier, Dami A %A Albecka, Anna %A Singh, Sujeet %A Pandey, Rajesh %A Brown, Jonathan %A Zhou, Jie %A Goonawardane, Niluka %A Mishra, Swapnil %A Whittaker, Charles %A Mellan, Thomas %A Marwal, Robin %A Datta, Meena %A Sengupta, Shantanu %A Ponnusamy, Kalaiarasan %A Radhakrishnan, Venkatraman Srinivasan %A Abdullahi, Adam %A Charles, Oscar %A Chattopadhyay, Partha %A Devi, Priti %A Caputo, Daniela %A Peacock, Tom %A Wattal, Chand %A Goel, Neeraj %A Satwik, Ambrish %A Vaishya, Raju %A Agarwal, Meenakshi %A Mavousian, Antranik %A Lee, Joo Hyeon %A Bassi, Jessica %A Silacci-Fegni, Chiara %A Saliba, Christian %A Pinto, Dora %A Irie, Takashi %A Yoshida, Isao %A Hamilton, William L %A Sato, Kei %A Bhatt, Samir %A Flaxman, Seth %A James, Leo C %A Corti, Davide %A Piccoli, Luca %A Barclay, Wendy S %A Rakshit, Partha %A Agrawal, Anurag %A Gupta, Ravindra K %K Antibodies, Neutralizing %K Cell Fusion %K Cell Line %K COVID-19 Vaccines %K Female %K Health Personnel %K Humans %K Immune Evasion %K India %K Kinetics %K Male %K SARS-CoV-2 %K Spike Glycoprotein, Coronavirus %K Vaccination %K Virus Replication %X

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

%B Nature %V 599 %P 114-119 %8 2021 11 %G eng %N 7883 %R 10.1038/s41586-021-03944-y %0 Journal Article %J Angew Chem Int Ed Engl %D 2020 %T A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase. %A Arya, Chetan Kumar %A Yadav, Swati %A Fine, Jonathan %A Casanal, Ana %A Chopra, Gaurav %A Ramanathan, Gurunath %A Vinothkumar, Kutti R %A Subramanian, Ramaswamy %X

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

%B Angew Chem Int Ed Engl %V 59 %P 16961-16966 %8 2020 09 21 %G eng %N 39 %R 10.1002/anie.202005332 %0 Journal Article %J Nat Commun %D 2020 %T The Rad53-Spt21 and Tel1 axes couple glucose tolerance to histone dosage and subtelomeric silencing. %A Bruhn, Christopher %A Ajazi, Arta %A Ferrari, Elisa %A Lanz, Michael Charles %A Batrin, Renaud %A Choudhary, Ramveer %A Walvekar, Adhish %A Laxman, Sunil %A Longhese, Maria Pia %A Fabre, Emmanuelle %A Smolka, Marcus Bustamente %A Foiani, Marco %K Acetylation %K Ataxia Telangiectasia Mutated Proteins %K Cell Cycle Proteins %K Checkpoint Kinase 2 %K DNA Damage %K DNA Repair %K Gene Silencing %K Glucose %K Histone Deacetylases %K Histones %K Intracellular Signaling Peptides and Proteins %K Mutation %K Phosphorylation %K Protein-Serine-Threonine Kinases %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Serine %K Telomere %K Transcription Factors %X

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21 on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1 and Rpd3 activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

%B Nat Commun %V 11 %P 4154 %8 2020 08 19 %G eng %N 1 %R 10.1038/s41467-020-17961-4 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2020 %T Stress-induced modulation of endocannabinoid signaling leads to delayed strengthening of synaptic connectivity in the amygdala. %A Yasmin, Farhana %A Colangeli, Roberto %A Morena, Maria %A Filipski, Sarah %A van der Stelt, Mario %A Pittman, Quentin J %A Hillard, Cecilia J %A Teskey, G Campbell %A McEwen, Bruce S %A Hill, Matthew N %A Chattarji, Sumantra %K Administration, Oral %K Amidohydrolases %K Animals %K Basolateral Nuclear Complex %K Cannabinoid Receptor Antagonists %K Disease Models, Animal %K Emotions %K Endocannabinoids %K Enzyme Inhibitors %K Excitatory Postsynaptic Potentials %K Humans %K Male %K Rats %K Receptor, Cannabinoid, CB1 %K Signal Transduction %K Stress, Psychological %X

Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CBR), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced -arachidonoylethanolamine (AEA), an endogenous ligand of CBR. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.

%B Proc Natl Acad Sci U S A %V 117 %P 650-655 %8 2020 01 07 %G eng %N 1 %R 10.1073/pnas.1910322116 %0 Journal Article %J Stem Cell Res %D 2019 %T Generation of two iPSC lines with either a heterozygous V717I or a heterozygous KM670/671NL mutation in the APP gene. %A Frederiksen, Henriette R %A Holst, Bjørn %A Ramakrishna, Sarayu %A Muddashetty, Ravi %A Schmid, Benjamin %A Freude, Kristine %X

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people worldwide. Mutations in the genes PSEN1, PSEN2 or APP are known to cause familial forms of AD with an early age of onset. In this study, specific pathogenic mutations in the APP gene were introduced into an iPSC line from a healthy individual by the use of CRISPR-Cas9. The study resulted in the generation of two new cell lines, one carrying the V717I APP mutation and one with the KM670/671NL APP mutation.

%B Stem Cell Res %V 34 %P 101368 %8 2019 Jan %G eng %R 10.1016/j.scr.2018.101368 %0 Journal Article %J PLoS One %D 2019 %T Graft-implanted, enzyme responsive, tacrolimus-eluting hydrogel enables long-term survival of orthotopic porcine limb vascularized composite allografts: A proof of concept study. %A Fries, C Anton %A Lawson, Shari D %A Wang, Lin C %A Slaughter, Kai V %A Vemula, Praveen K %A Dhayani, Ashish %A Joshi, Nitin %A Karp, Jeffrey M %A Rickard, Rory F %A Gorantla, Vijay S %A Davis, Michael R %X

BACKGROUND: Currently, patients receiving vascularized composite allotransplantation (VCA) grafts must take long-term systemic immunosuppressive therapy to prevent immunologic rejection. The morbidity and mortality associated with these medications is the single greatest barrier to more patients being able to receive these life-enhancing transplants. In contrast to solid organs, VCA, exemplified by hand or face transplants, allow visual diagnosis of clinical acute rejection (AR), directed biopsy and targeted graft therapies. Local immunosuppression in VCA could reduce systemic drug exposure and limit adverse effects. This proof of concept study evaluated, in a large animal forelimb VCA model, the efficacy and tolerability of a novel graft-implanted enzyme-responsive, tacrolimus (TAC)-eluting hydrogel platform, in achieving long-term graft survival.

METHODS: Orthotopic forelimb VCA were performed in single haplotype mismatched mini-swine. Controls (n = 2) received no treatment. Two groups received TAC hydrogel: high dose (n = 4, 91 mg TAC) and low dose (n = 4, 49 mg TAC). The goal was to find a dose that was tolerable and resulted in long-term graft survival. Limbs were evaluated for clinical and histopathological signs of AR. TAC levels were measured in serial blood and skin tissue samples. Tolerability of the dose was evaluated by monitoring animal feeding behavior and weight.

RESULTS: Control limbs underwent Banff Grade IV AR by post-operative day six. Low dose TAC hydrogel treatment resulted in long-term graft survival time to onset of Grade IV AR ranging from 56 days to 93 days. High dose TAC hydrogel also resulted in long-term graft survival (24 to 42 days), but was not well tolerated.

CONCLUSION: Graft-implanted TAC-loaded hydrogel delays the onset of Grade IV AR of mismatched porcine forelimb VCA grafts, resulting in long term graft survival and demonstrates dose-dependent tolerability.

%B PLoS One %V 14 %P e0210914 %8 2019 %G eng %N 1 %R 10.1371/journal.pone.0210914 %0 Journal Article %J Microb Biotechnol %D 2018 %T Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. %A Bairy, Sneha %A Gopalan, Lakshmi Narayanan %A Setty, Thanuja Gangi %A Srinivasachari, Sathya %A Manjunath, Lavanyaa %A Kumar, Jay Prakash %A Guntupalli, Sai R %A Bose, Sucharita %A Nayak, Vinod %A Ghosh, Swagatha %A Sathyanarayanan, Nitish %A Caing-Carlsson, Rhawnie %A Wahlgren, Weixiao Yuan %A Friemann, Rosmarie %A Ramaswamy, S %A Neerathilingam, Muniasamy %X

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

%B Microb Biotechnol %V 11 %P 420-428 %8 2018 Mar %G eng %N 2 %R 10.1111/1751-7915.13041 %0 Journal Article %J Biophys Rev %D 2018 %T "Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes. %A North, Rachel A %A Horne, Christopher R %A Davies, James S %A Remus, Daniela M %A Muscroft-Taylor, Andrew C %A Goyal, Parveen %A Wahlgren, Weixiao Yuan %A Ramaswamy, S %A Friemann, Rosmarie %A Dobson, Renwick C J %X

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

%B Biophys Rev %V 10 %P 219-227 %8 2018 Apr %G eng %N 2 %R 10.1007/s12551-017-0343-x %0 Journal Article %J Front Chem %D 2018 %T The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity. %A North, Rachel A %A Wahlgren, Weixiao Y %A Remus, Daniela M %A Scalise, Mariafrancesca %A Kessans, Sarah A %A Dunevall, Elin %A Claesson, Elin %A Soares da Costa, Tatiana P %A Perugini, Matthew A %A Ramaswamy, S %A Allison, Jane R %A Indiveri, Cesare %A Friemann, Rosmarie %A Dobson, Renwick C J %X

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from (SiaT). We demonstrate that SiaT rescues an strain lacking its endogenous sialic acid transporter when grown on the sialic acids -acetylneuraminic acid (Neu5Ac) or -glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SiaT that may explain the altered specificity. SiaT is shown to be electrogenic, and transport is dependent upon more than one Na ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SiaT, and developing the and assay systems, our work underpins the design of inhibitors to this transporter.

%B Front Chem %V 6 %P 233 %8 2018 %G eng %R 10.3389/fchem.2018.00233 %0 Journal Article %J Nat Commun %D 2018 %T Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site. %A Wahlgren, Weixiao Y %A Dunevall, Elin %A North, Rachel A %A Paz, Aviv %A Scalise, Mariafrancesca %A Bisignano, Paola %A Bengtsson-Palme, Johan %A Goyal, Parveen %A Claesson, Elin %A Caing-Carlsson, Rhawnie %A Andersson, Rebecka %A Beis, Konstantinos %A Nilsson, Ulf J %A Farewell, Anne %A Pochini, Lorena %A Indiveri, Cesare %A Grabe, Michael %A Dobson, Renwick C J %A Abramson, Jeff %A Ramaswamy, S %A Friemann, Rosmarie %X

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na ions. One Na binds to the conserved Na2 site, while the second Na binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na sites regulate N-acetylneuraminic acid transport.

%B Nat Commun %V 9 %P 1753 %8 2018 May 01 %G eng %N 1 %R 10.1038/s41467-018-04045-7 %0 Journal Article %J Acta Crystallogr F Struct Biol Commun %D 2017 %T Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. %A Caing-Carlsson, Rhawnie %A Goyal, Parveen %A Sharma, Amit %A Ghosh, Swagatha %A Setty, Thanuja Gangi %A North, Rachel A %A Friemann, Rosmarie %A Ramaswamy, S %K Adenosine Triphosphate %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Cloning, Molecular %K Crystallography, X-Ray %K Escherichia coli %K Fusobacterium nucleatum %K Gene Expression %K Genetic Vectors %K Hexosamines %K Models, Molecular %K Phosphotransferases (Alcohol Group Acceptor) %K Protein Binding %K Protein Conformation, alpha-Helical %K Protein Conformation, beta-Strand %K Protein Interaction Domains and Motifs %K Protein Multimerization %K Recombinant Proteins %K Sequence Alignment %K Sequence Homology, Amino Acid %K Substrate Specificity %X

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

%B Acta Crystallogr F Struct Biol Commun %V 73 %P 356-362 %8 2017 Jun 01 %G eng %N Pt 6 %R 10.1107/S2053230X17007439 %0 Journal Article %J Biochemistry %D 2017 %T Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. %A Plapp, Bryce V %A Savarimuthu, Baskar Raj %A Ferraro, Daniel J %A Rubach, Jon K %A Brown, Eric N %A Ramaswamy, S %K 2,2'-Dipyridyl %K Adenosine Diphosphate Ribose %K Alcohol Dehydrogenase %K Animals %K Catalytic Domain %K Crystallography, X-Ray %K Formamides %K Horses %K Kinetics %K Liver %K Models, Molecular %K NAD %K Phenanthrolines %K Protein Binding %K Protein Conformation %K Water %K Zinc %X

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

%B Biochemistry %V 56 %P 3632-3646 %8 2017 07 18 %G eng %N 28 %R 10.1021/acs.biochem.7b00446 %0 Journal Article %J IUCrJ %D 2017 %T {One enzyme, many reactions: structural basis for the various reactions catalyzed by naphthalene 1,2-dioxygenase} %A Ferraro, Daniel J. %A Okerlund, Adam %A Brown, Eric %A Ramaswamy, S. %K 2-dioxygenase %K deoxygenation %K monooxygenation %K naphthalene 1 %K substrate orientation %K sulfoxidation %X

Rieske nonheme iron oxygenases (ROs) are a well studied class of enzymes. Naphthalene 1,2-dioxygenase (NDO) is used as a model to study ROs. Previous work has shown how side-on binding of oxygen to the mononuclear iron provides this enzyme with the ability to catalyze stereospecific and regiospecific ıt cis}-dihydroxylation reactions. It has been well documented that ROs catalyze a variety of other reactions, including mono-oxygenation, desaturation, O- and N-dealkylation, sulfoxidation ıt etc}. NDO itself catalyzes a variety of these reactions. Structures of NDO in complex with a number of different substrates show that the orientation of the substrate in the active site controls not only the regiospecificity and stereospecificity, but also the type of reaction catalyzed. It is proposed that the mononuclear iron-activated dioxygen attacks the atoms of the substrate that are most proximal to it. The promiscuity of delivering two products (apparently by two different reactions) from the same substrate can be explained by the possible binding of the substrate in slightly different orientations aided by the observed flexibility of residues in the binding pocket.

%B IUCrJ %V 4 %P 648–656 %8 Sep %G eng %U https://doi.org/10.1107/S2052252517008223 %R 10.1107/S2052252517008223 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2016 %T Blue protein with red fluorescence. %A Ghosh, Swagatha %A Yu, Chi-Li %A Ferraro, Daniel J %A Sudha, Sai %A Pal, Samir Kumar %A Schaefer, Wayne F %A Gibson, David T %A Ramaswamy, S %K Biliverdine %K Crystallography, X-Ray %K Fluorescence %K Models, Molecular %K Proteins %K Recombinant Proteins %X

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

%B Proc Natl Acad Sci U S A %V 113 %P 11513-11518 %8 2016 10 11 %G eng %N 41 %R 10.1073/pnas.1525622113