Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies. Here, we report the synthesis of a mimic of ADP-ribosylated peptide derived from histone H2B that contains carba-ADP-ribosylated glutamine as a potential mimic for Glu-ADPr. We synthesized a cyclopentitol-ribofuranosyl derivative of 5'-phosphoribosylated Fmoc-glutamine and used this in the solid-phase synthesis of the carba-ADPr-peptide mimicking the ADP-ribosylated N-terminal tail of histone H2B. Binding studies with isothermal calorimetry demonstrate that the macrodomains of human MacroD2 and TARG1 bind to carba-ADPr-peptide in the same way as ADPr-peptides containing the native ADP-riboside moiety connected to the side chain of glutamine in the same peptide sequence.

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

Homology modeling and molecular docking studies to decrease glutamine affinity of Yarrowia lipolytica L-asparaginase.

L-Asparaginase is a key component in the treatment of leukemias and lymphomas. However, the glutamine affinity of this therapeutic enzyme is an off-target activity that causes several side effects. The modeling and molecular docking study of Yarrowia lipolytica L-asparaginase (YL-ASNase) to reduce its l-glutamine affinity and increase its stability was the aim of this study. Protein-ligand interactions of wild-type and different mutants of YL-ASNase against L-asparagine compared to l-glutamine were assessed using AutoDock Vina tools because the crystal structure of YL-ASNase does not exist in the protein data banks. The results showed that three mutants, T171S, T171S-N60A, and T171A-T223A, caused a considerable increase in L-asparagine affinity and a decrease in l-glutamine affinity as compared to the wild-type and other mutants. Then, molecular dynamics simulation and MM/GBSA free energy were applied to assess the stability of protein structure and its interaction with ligands. The three mutated proteins, especially T171S-N60A, had higher stability and interactions with L-asparagine than l-glutamine in comparison with the wild-type. The YL-ASNase mutants could be introduced as appropriate therapeutic candidates that might cause lower side effects. However, the functional properties of these mutated enzymes need to be confirmed by genetic manipulation and in vitro and in vivo studies.

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Aminoacylase efficiently hydrolyses fatty acid amino acid conjugates of Helicoverpa armigera potentially to increase the pool of glutamine.

One of the most prevalent bioactive molecules present in the oral secretion (OS) of lepidopteran insects is fatty acid amino acid conjugates (FACs). Insect dietary components have influence on the synthesis and retaining the pool of FACs in the OS. We noted differential and diet-specific accumulation of FACs in the OS of Helicoverpa armigera by using Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry. Interestingly, we identified FACs hydrolyzing enzyme aminoacylase (HaACY) in the OS of H. armigera through proteomic analysis. Next, we have cloned, expressed, and purified active recombinant HaACY in the bacterial system. Recombinant HaACY hydrolyzes all the six identified FACs in the OS of H. armigera larvae fed on host and non-host plants and releases respective fatty acid and glutamine. In these six FACs, fatty acid moieties vary while amino acid glutamine was common. Glutamine obtained upon hydrolysis of FACs by HaACY might serve as an amino acid pool for insect growth and development. To understand the substrate choices of HaACY, we chemically synthesized, purified, and characterized all the six FACs. Interestingly, rHaACY also shows hydrolysis of synthetic FACs into respective fatty acid and glutamine. Our results underline the importance of diet on accumulation of FACs and role of aminoacylase(s) in regulating the level of FACs and glutamine.

M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

Assessing structural insights into in-house arylsulfonyl L-(+) glutamine MMP-2 inhibitors as promising anticancer agents through structure-based computational modelling approaches.

MMP-2 is potentially contributing to several cancer progressions including leukaemias. Therefore, considering MMP-2 as a promising target, novel anticancer compounds may be designed. Here, 32 in-house arylsulfonyl L-(+) glutamines were subjected to various structure-based computational modelling approaches to recognize crucial structural attributes along with the spatial orientation for higher MMP-2 inhibition. Again, the docking-based 2D-QSAR study revealed that the Coulomb energy conferred by Tyr142 and total interaction energy conferred by Ala84 was crucial for MMP-2 inhibition. Importantly, the docking-dependent CoMFA and CoMSIA study revealed the importance of favourable steric, electrostatic, and hydrophobic substituents at the terminal phenyl ring. The MD simulation study revealed a lower fluctuation in the RMSD, RMSF, and Rg values indicating stable binding interactions of MMP-2 and these molecules. Moreover, the residual hydrogen bond and their interaction analysis disclosed crucial amino acid residues responsible for forming potential hydrogen bonding for higher MMP-2 inhibition. The results can effectively aid in the design and discovery of promising small-molecule drug-like MMP-2 inhibitors with greater anticancer potential in the future.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures.

We apply an integrated approach combining microsecond MD simulations and (polarizable) QM/MM calculations of NMR, FTIR, and UV-vis spectra to validate the structure of the light-activated form of the AppA photoreceptor, an example of blue light using flavin (BLUF) protein domain. The latter photoactivate through a proton-coupled electron transfer (PCET) that results in a tautomerization of a conserved glutamine residue in the active site, but this mechanism has never been spectroscopically proven for AppA, which has been always considered as an exception. Our simulations instead confirm that the spectral features observed upon AppA photoactivation are indeed directly connected to the tautomer form of glutamine as predicted by the PCET mechanism. In addition, we observe small but significant changes in the AppA structure, which are transmitted from the flavin binding pocket to the surface of the protein.

New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

A Toolbox for Glutamine Use in Dissolution Dynamic Nuclear Polarization: from Enzymatic Reaction Monitoring to the Study of Cellular Metabolic Pathways and Imaging.

Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E. coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

Evaluation of Glutaminolysis in T Cells.

The activity of living cells is necessarily dependent on the amount of available bioenergy. In T cells, the latter is mainly derived from ATP, a molecular energy "coin" generated by one of several metabolic processes that differ in their ability to satisfy energy demand. Thus, whereas naïve or quiescent T cells efficiently utilize oxidative phosphorylation to generate ATP, T cells subjected to antigenic stimulation followed by clonal expansion and cytokine production meet their increased need for energy by supplementing ATP generation by oxidative phosphorylation with ATP generation by glycolysis. Yet additional need for ATP can be met by other basic biologic sources of energy such as glutamine, an amino acid that is metabolized through a process called glutaminolysis to result in end products that flows into the TCA cycle and augment ATP generation by oxidative phosphorylation. It is now possible to track the dominant energy supplying processes (i.e., the ATP generation process) in differentiating or activated T cells in a real-time manner. Here, we provide one element of such tracking by describing protocols for the assessment of the contribution of glutaminolysis to overall ATP production within different T cell subsets. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Evaluation of the role of glutaminolysis during T cell activation/differentiation Basic Protocol 2: Evaluation of the role of glutaminolysis in T cell responses utilizing glutaminolysis inhibitors Basic Protocol 3: Evaluation of the effect of glutaminolysis on cellular oxidative phosphorylation/glycolysis.

PET Imaging of Active Invasive Fungal Infections with d-[5-11C]-Glutamine.

The increasing prevalence and severity of invasive fungal infections (IFIs), especially in immunocompromised populations, has amplified the need for rapid diagnosis of fungal pathogens. Radiotracers derived from d-amino acids (DAAs) show promise as bacterial-specific positron emission tomography (PET) imaging agents due to their preferential consumption by bacteria and largely nonutilization by hosts. Unlike mammals, fungi can utilize external DAAs including d-glutamine for their growth by rapidly upregulating DAA oxidases. Additionally, glutamine is essential for fungal nitrogen assimilation, survival, and virulence. We previously validated d-[5-11C]-glutamine (d-[5-11C]-Gln) as an efficient radiotracer targeting live bacterial soft-tissue infections. Here, we further expanded this investigation to evaluate its translational potential for PET imaging of IFIs in immunocompetent mouse models subcutaneously (SubQ) and intramuscularly (IM) infected with Candida albicans (C. albicans), using its l-isomer counterpart (l-[5-11C]-Gln) as a control. Comparative studies between pathogens showed significantly (p < 0.05) higher uptake in fungi (C. albicans and C. tropicalis) versus tested bacterial species for d-[5-11C]-Gln, suggesting that it could potentially serve as a more sensitive radiotracer for detection of fungal infections. Additionally, comparative PET imaging studies in immunocompetent infected mice demonstrated significantly higher infection-to-background ratios for d- versus l-[5-11C]-Gln in both SubQ (ratio = 1.97, p = 0.043) and IM (ratio = 1.97, p = 0.028) infections. Fungal infection imaging specificity was confirmed with no significant difference observed between localized inflammation sites versus untreated muscle background (heat-killed injection site/untreated muscle: ∼1.1). Taken together, this work demonstrates the translational potential of d-[5-11C]-Gln for noninvasive PET imaging of IFIs.

Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine.

In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

[HEMK-Like Methyltransferases in the Regulation of Cellular Processes].

Human translational methyltransferase (methylase) HEMK2, whose orthologues are found in many prokaryotes and eukaryotes, methylates such diverse substrates as glutamine and lysine residues in proteins, deoxyadenosine in DNA, and arsenicals. One of the important substrate of HEMK2 methylase is a glutamine residue in the GGQ ultra-conservative motif of the eukaryotic release factor 1 (eRF1). Release factor methylation by HEMK2 orthologs is conserved among eukaryotes, archaea, and bacteria, although bacterial release factors differ in sequence and structure from eukaryotic ones. In this review, we consider the features of human HEMK2 methylase and its orthologs as multifunctional enzymes that regulate cellular processes, in particular, protein biosynthesis.

Deciphering the Spectral Tuning Mechanism in Proteorhodopsin: The Dominant Role of Electrostatics Instead of Chromophore Geometry.

Proteorhodopsin (PR) is a photoactive proton pump found in marine bacteria. There are two phenotypes of PR exhibiting an environmental adaptation to the ocean's depth which tunes their maximum absorption: blue-absorbing proteorhodopsin (BPR) and green-absorbing proteorhodopsin (GPR). This blue/green color-shift is controlled by a glutamine to leucine substitution at position 105 which accounts for a 20 nm shift. Typically, spectral tuning in rhodopsins is rationalized by the external point charge model but the Q105L mutation is charge neutral. To study this tuning mechanism, we employed the hybrid QM/MM method with sampling from molecular dynamics. Our results reveal that the positive partial charge of glutamine near the C14 -C15 bond of retinal shortens the effective conjugation length of the chromophore compared to the leucine residue. The derived mechanism can be applied to explain the color regulation in other retinal proteins and can serve as a guideline for rational design of spectral shifts.

Physiological synaptic activity and recognition memory require astroglial glutamine.

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.

High-resolution structures of mitochondrial glutaminase C tetramers indicate conformational changes upon phosphate binding.

The mitochondrial enzyme glutaminase C (GAC) is upregulated in many cancer cells to catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. The dependence of cancer cells on this transformed metabolic pathway highlights GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescent reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the Y466W GAC tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric Y466W GAC bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e., the "lid"), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates, resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the Y466W GAC tetramer exhibited bound glutamine with closed lids. Glutamine can bind with high affinity to each subunit, which subsequently undergo simultaneous catalysis. These findings explain how the regulated transitioning of GAC between different conformational states ensures that maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.

Mass spectrometric analysis of protein deamidation - A focus on top-down and middle-down mass spectrometry.

Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been demonstrated to be associated with protein folding, enzymatic activity, and degradation, as well as aging, cancer, and neurodegenerative diseases. To gain a better understanding on the biological roles of protein deamidation in aging and diseases, mass spectrometry (MS) has been employed in the identification of deamidated protein species and comprehensive characterization of deamidation sites. Three main MS approaches, top-down, middle-down, and bottom-up have been applied in the study of protein deamidation with high sensitivity, throughput, and accuracy. In this review, we discuss the application of top-down and middle-down MS in the study of protein deamidation, including sample preparation methods, separation strategies, MS and MS/MS techniques and data analysis. The advantages and drawbacks of these two approaches are also discussed and compared with those of the bottom-up method. The development of top-down and middle-down MS methods provides new strategies for protein deamidation analysis and gives new insights into the biological significance of protein deamidation in diseases.