%0 Journal Article %J Mol Neurobiol %D 2022 %T Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis. %A D'Souza, Michelle Ninochka %A Ramakrishna, Sarayu %A Radhakrishna, Bindushree K %A Jhaveri, Vishwaja %A Ravindran, Sreenath %A Yeramala, Lahari %A Nair, Deepak %A Palakodeti, Dasaradhi %A Muddashetty, Ravi S %K Fragile X Mental Retardation Protein %K Fragile X Syndrome %K Humans %K Microtubules %K Neurons %K Protein Biosynthesis %K Ribosomes %K RNA, Messenger %X

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP's domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP's domains in translation regulation.

%B Mol Neurobiol %V 59 %P 7370-7392 %8 2022 Dec %G eng %N 12 %R 10.1007/s12035-022-03049-1 %0 Journal Article %J RSC Adv %D 2022 %T Modulation of biliverdin dynamics and spectral properties by Sandercyanin. %A Ghosh, Swagatha %A Mondal, Sayan %A Yadav, Keerti %A Aggarwal, Shantanu %A Schaefer, Wayne F %A Narayana, Chandrabhas %A Subramanian, Ramaswamy %X

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

%B RSC Adv %V 12 %P 20296-20304 %8 2022 Jul 06 %G eng %N 31 %R 10.1039/d2ra02880h %0 Journal Article %J NPJ Genom Med %D 2022 %T Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy. %A Lesurf, Robert %A Said, Abdelrahman %A Akinrinade, Oyediran %A Breckpot, Jeroen %A Delfosse, Kathleen %A Liu, Ting %A Yao, Roderick %A Persad, Gabrielle %A McKenna, Fintan %A Noche, Ramil R %A Oliveros, Winona %A Mattioli, Kaia %A Shah, Shreya %A Miron, Anastasia %A Yang, Qian %A Meng, Guoliang %A Yue, Michelle Chan Seng %A Sung, Wilson W L %A Thiruvahindrapuram, Bhooma %A Lougheed, Jane %A Oechslin, Erwin %A Mondal, Tapas %A Bergin, Lynn %A Smythe, John %A Jayappa, Shashank %A Rao, Vinay J %A Shenthar, Jayaprakash %A Dhandapany, Perundurai S %A Semsarian, Christopher %A Weintraub, Robert G %A Bagnall, Richard D %A Ingles, Jodie %A Melé, Marta %A Maass, Philipp G %A Ellis, James %A Scherer, Stephen W %A Mital, Seema %X

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

%B NPJ Genom Med %V 7 %P 18 %8 2022 Mar 14 %G eng %N 1 %R 10.1038/s41525-022-00288-y %0 Journal Article %J Biophys J %D 2021 %T Biophysical properties of the isolated spike protein binding helix of human ACE2. %A Das, Anirban %A Vishvakarma, Vicky %A Dey, Arpan %A Dey, Simli %A Gupta, Ankur %A Das, Mitradip %A Vishwakarma, Krishna Kant %A Roy, Debsankar Saha %A Yadav, Swati %A Kesarwani, Shubham %A Venkatramani, Ravindra %A Maiti, Sudipta %K Angiotensin-Converting Enzyme 2 %K COVID-19 %K Humans %K Peptidyl-Dipeptidase A %K Protein Binding %K SARS-CoV-2 %K Spike Glycoprotein, Coronavirus %X

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel β-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

%B Biophys J %V 120 %P 2785-2792 %8 2021 07 20 %G eng %N 14 %R 10.1016/j.bpj.2021.06.017 %0 Journal Article %J Elife %D 2021 %T Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in tracheoblasts. %A Kizhedathu, Amrutha %A Chhajed, Piyush %A Yeramala, Lahari %A Sain Basu, Deblina %A Mukherjee, Tina %A Vinothkumar, Kutti R %A Guha, Arjun %X

Progenitors of the thoracic tracheal system of adult (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an HO generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/ Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of HO in minutes. The findings presented reveal that HO activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

%B Elife %V 10 %8 2021 Oct 08 %G eng %R 10.7554/eLife.68636 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2021 %T A regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes. %A Nikhat, Sameena %A Yadavalli, Anurupa D %A Prusty, Arpita %A Narayan, Priyanka K %A Palakodeti, Dasaradhi %A Murre, Cornelis %A Pongubala, Jagan M R %K Animals %K B-Lymphocytes %K Cell Differentiation %K Cell Lineage %K Gene Expression %K Gene Expression Profiling %K Gene Regulatory Networks %K Hematopoietic Stem Cells %K Mice %K MicroRNAs %K Myeloid Cells %K T-Lymphocytes %X

The commitment of hematopoietic multipotent progenitors (MPPs) toward a particular lineage involves activation of cell type-specific genes and silencing of genes that promote alternate cell fates. Although the gene expression programs of early-B and early-T lymphocyte development are mutually exclusive, we show that these cell types exhibit significantly correlated microRNA (miRNA) profiles. However, their corresponding miRNA targetomes are distinct and predominated by transcripts associated with natural killer, dendritic cell, and myeloid lineages, suggesting that miRNAs function in a cell-autonomous manner. The combinatorial expression of miRNAs miR-186-5p, miR-128-3p, and miR-330-5p in MPPs significantly attenuates their myeloid differentiation potential due to repression of myeloid-associated transcripts. Depletion of these miRNAs caused a pronounced de-repression of myeloid lineage targets in differentiating early-B and early-T cells, resulting in a mixed-lineage gene expression pattern. De novo motif analysis combined with an assay of promoter activities indicates that B as well as T lineage determinants drive the expression of these miRNAs in lymphoid lineages. Collectively, we present a paradigm that miRNAs are conserved between developing B and T lymphocytes, yet they target distinct sets of promiscuously expressed lineage-inappropriate genes to suppress the alternate cell-fate options. Thus, our studies provide a comprehensive compendium of miRNAs with functional implications for B and T lymphocyte development.

%B Proc Natl Acad Sci U S A %V 118 %8 2021 11 16 %G eng %N 46 %R 10.1073/pnas.2104297118 %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 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 EMBO Rep %D 2019 %T Dynamic expression of tRNA-derived small RNAs define cellular states. %A Krishna, Srikar %A Yim, Daniel Gr %A Lakshmanan, Vairavan %A Tirumalai, Varsha %A Koh, Judice Ly %A Park, Jung Eun %A Cheong, Jit Kong %A Low, Joo Leng %A Lim, Michelle Js %A Sze, Siu Kwan %A Shivaprasad, Padubidri %A Gulyani, Akash %A Raghavan, Srikala %A Palakodeti, Dasaradhi %A DasGupta, Ramanuj %X

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.

%B EMBO Rep %V 20 %P e47789 %8 2019 Jul %G eng %N 7 %R 10.15252/embr.201947789 %0 Journal Article %J Nat Commun %D 2018 %T Towards an arthritis flare-responsive drug delivery system. %A Joshi, Nitin %A Yan, Jing %A Levy, Seth %A Bhagchandani, Sachin %A Slaughter, Kai V %A Sherman, Nicholas E %A Amirault, Julian %A Wang, Yufeng %A Riegel, Logan %A He, Xueyin %A Rui, Tan Shi %A Valic, Michael %A Vemula, Praveen K %A Miranda, Oscar R %A Levy, Oren %A Gravallese, Ellen M %A Aliprantis, Antonios O %A Ermann, Joerg %A Karp, Jeffrey M %X

Local delivery of therapeutics for the treatment of inflammatory arthritis (IA) is limited by short intra-articular half-lives. Since IA severity often fluctuates over time, a local drug delivery method that titrates drug release to arthritis activity would represent an attractive paradigm in IA therapy. Here we report the development of a hydrogel platform that exhibits disassembly and drug release controlled by the concentration of enzymes expressed during arthritis flares. In vitro, hydrogel loaded with triamcinolone acetonide (TA) releases drug on-demand upon exposure to enzymes or synovial fluid from patients with rheumatoid arthritis. In arthritic mice, hydrogel loaded with a fluorescent dye demonstrates flare-dependent disassembly measured as loss of fluorescence. Moreover, a single dose of TA-loaded hydrogel but not the equivalent dose of locally injected free TA reduces arthritis activity in the injected paw. Together, our data suggest flare-responsive hydrogel as a promising next-generation drug delivery approach for the treatment of IA.

%B Nat Commun %V 9 %P 1275 %8 2018 Apr 03 %G eng %N 1 %R 10.1038/s41467-018-03691-1 %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 %0 Journal Article %J IUCrJ %D 2016 %T Structure of a heterogeneous, glycosylated, lipid-bound, ıt in vivo-grown protein crystal at atomic resolution from the viviparous cockroach ıt Diploptera punctata %A Banerjee, Sanchari %A Coussens, Nathan P. %A Gallat, François-Xavier %A Sathyanarayanan, Nitish %A Srikanth, Jandhyam %A Yagi, Koichiro J. %A Gray, James S. S. %A Tobe, Stephen S. %A Stay, Barbara %A Chavas, Leonard M. G. %A Ramaswamy, Subramanian %K glycosylation %K protein heterogeneity %K sulfur-SAD %K viviparity in cockroach %X

Macromolecular crystals for X-ray diffraction studies are typically grown ıt in vitro} from pure and homogeneous samples; however, there are examples of protein crystals that have been identified ıt in vivo}. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown ıt in cellulo}. Here, an atomic resolution (1.2{\AA}) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These ıt in vivo}-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, ıt Diploptera punctata}. The milk proteins crystallized in space group ıt P}1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single ıt in vivo}-grown crystal of a natural protein in its native environment at atomic resolution.

%B IUCrJ %V 3 %P 282–293 %8 Jul %G eng %U http://dx.doi.org/10.1107/S2052252516008903 %R 10.1107/S2052252516008903