Asparagine Residues Research Articles

Front Cover: Characterization of Asx Turn Types and Their Connate Relationship with β‐Turns (Chem. Eur. J. 25/2022)

Bridging the gap between crystallized proteins and gas-phase models sheds light on the structure and the function of Asx turns. A comparison of the isolated Asx turns, based on an asparagine residue, observed in model peptides by using laser spectroscopic and quantum chemistry diagnostics, with the structures of crystallized proteins supports the premise that Asx turns foster or stabilize classical β-turn structures, and even promote β-bulges when Asn is followed by a Gly residue. More information can be found in the Research Article by D. J. Aitken, M. Mons et al. (DOI: 10.1002/chem.202104328).

Trends in deamidation across archaeological bones, ceramics and dental calculus.

The accumulation of post-translational modifications (PTMs) in proteins throughout the lifecycle has been studied for decades, particularly more so with the advent of soft-ionization mass spectrometry-based proteomic techniques. However, particular PTMs, such as the deamidations of asparagine and glutamine residues, continue to accumulate in proteins that remain into the forensic, archaeological, and palaeontological records. The accurate measurement of these ancient 'molecular timers' has been proposed as a method to not only differentiate between exogenous and endogenous proteins within complex mixtures (i.e., contamination), but also as a method of providing relative age estimations into geological time. In this study we explored the extent to which deamidation varies with chronological age across different proteins in bones, as well as investigated differences between proteins across dental calculus and archaeological ceramics. We also analysed the relationships between the observed extent of deamidation and the protein primary structure. We found that collagen obtained from archaeological bones showed a chronological dependence on the extent of deamidation observed, but only when they were from similar environments, supporting prior suggestions about 'thermal age' being a major influence on the deamidation observed. Our study on non-collagenous proteins (NCPs) in archaeological bones showed that while biglycan, and to a lesser extent chondroadherin, showed positive correlations between geological age and the extent of deamidation, others including fetuin-A and serum albumin did not. However, despite the well-known dependence of deamidation on the three-dimensional structure of the peptides, we were unable to find any clear correlation between the structural motifs of the peptides in archaeological bones and the extent of deamidation observed. Our analysis of a set of food proteins obtained from Neolithic archaeological ceramics in Çatalhöyük also showed similar deamidation levels irrespective of the protein structure. Overall, our results suggest that deamidation in archaeological samples could be useful for obtaining additional information beyond identification of species and tissue type, be that as a measure of protein endogeneity and potential contamination, or a measure of protein degradation, or as an indicator of thermal age and for relative dating; however, further research needs to be undertaken to understand why particular proteins are better for this than others, going beyond simple consideration of their secondary structure.

Importance of Asparagine 202 in Manipulating Active Site Structure and Substrate Preference for Human CYP17A1.

The multifunctional cytochrome P450 17A1 (CYP17A1) plays a crucial role in human steroid hormone synthesis (UniProtKB─P05093). It first carries out standard monooxygenase chemistry, converting pregnenolone (PREG) and progesterone (PROG) into 17OH-PREG and 17OH-PROG, utilizing a "Compound I" to initiate hydrogen abstraction and radical recombination in the classic "oxygen rebound" mechanism. Additionally, these hydroxylated products also serve as substrates in a second oxidative cycle which cleaves the 17-20 carbon-carbon bond to form dehydroepiandrosterone and androstenedione, which are key precursors in the generation of powerful androgens and estrogens. Interestingly, in humans, with 17OH-PREG, this so-called lyase reaction is more efficient than with 17OH-PROG, based on Kcat/Km values. In the present work, the asparagine residue at 202 position was replaced by serine, an alteration which can affect substrate orientation and control substrate preference for the lyase reaction. First, we report studies of solvent isotope effects for the N202S CYP17A1 mutant in the presence of 17OH-PREG and 17OH-PROG, which suggest that the ferric peroxo species is the predominant catalytically active intermediate in the lyase step. This conclusion is further supported by employing a combination of cryoradiolysis and resonance Raman techniques to successfully trap and structurally characterize the key reaction intermediates, including the peroxo, the hydroperoxo, and the crucial peroxo-hemiketal intermediate. Collectively, these studies show that the mutation causes active site structural changes that alter the H-bonding interactions with the key Fe-O-O fragment and the degree of protonation of the reactive ferric peroxo intermediate, thereby impacting lyase efficiency.

The flocculant Saccharomyces cerevisiae strain gains robustness via alteration of the cell wall hydrophobicity

When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence

BackgroundVerticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown.ResultsNine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type.ConclusionsIn the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Abstract P3-14-13: Metabolic links to socioeconomic stresses uniquely affecting race in normal breast tissue at risk for breast cancer

Abstract A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is not incidence, but aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. There is ongoing debate regarding whether the underlying cause of higher mortality is related to healthcare inequalities or due to ancestry dependent molecular features found in normal breast tissue that facilitate the aggressive phenotype found in black women. One hypothesis is that chronic social and economic stressors result in molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue, and these chronic stresses differ by race/ethnicity. In this study, we investigate molecular pattern changes in tissue N-glycosylation in a cohort of ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, was tested against social stressors. Social stresses included marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (WB=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. There was no significant difference in age (Median age BW 42.5, 95% CI [40.0, 45.0]; WW 42.0, 95% CI [39.7, 44.3]). A subgroup of women had similar BMI (BW, n=24 Median BMI 29.5, 95% CI [27.6, 32.3]; WW, n=24 28.3, 95% CI [23.5, 31.6]. BW women had an overall lower median risk by 5-year Gail score, which, (BW 9.4, 95% CI [8.9, 9.9]; WW 10.4, 95% CI [8.1, 12.6]). Area under the receiver operating curve (AUC) ≥0.70, Brown/Wilson p-value <0.001 was used to assess for individual glycosylation differences in normal breast tissue at risk for breast cancer. Out of 55 N-glycans profiled in normal breast by glycomics mass spectrometry, one N-glycan appeared dependent on ancestry with high sensitivity and specificity (AUC 0.788, Brown/Wilson p-value<0.0001). For women of the same BMI, a total of 12 N-glycans could report potential ancestry-dependent differences. Interestingly, when we fit a linear regression model with ancestry as group variable and socioeconomic covariates as predictors, different N-glycans associated with different socioeconomic stresses. Five N-glycans in particular linked to different socioeconomic stresses for both BW and WW. For white women, household income was strongly associated to certain N-glycans, while for black women, marriage status (married and single) was strongly associated with the same N-glycan signature. Current work focuses on understanding if combined N-glycan biosignatures can further help understand normal breast tissue at risk. The data suggests that metabolic patterns linked to socioeconomic stresses may contribute to breast cancer risk dependent on ancestry. This study lays the foundation for understanding the complexities linking socioeconomic stresses and molecular factors to their role in ancestry dependent breast cancer risk and aggressiveness in black women. Citation Format: Peggi M Angel, Yeonhee Park, Danielle A Scott, Denys Rujchanarong, Sean Brown, Richard R Drake, George E Sandusky, Harikrishna Nakshatri. Metabolic links to socioeconomic stresses uniquely affecting race in normal breast tissue at risk for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-13.

POS-337 ASPARAGINYL ENDOPEPTIDASE PROTECTS AGAINST PODOCYTE INJURY IN DIABETIC NEPHROPATHY THROUGH CLEAVING COFILIN-1

Asparaginyl endopeptidase (AEP) is a lysosomal cysteine protease that specifically cleaves its substrates after asparagine residues. Physiologically, AEP is most abundant in kidney proximal tubule and is required for normal kidney physiology and homeostasis. Recent studies reported that AEP exerts anti-fibrotic effect through degrading of matrix fibronection during unilateral ureteral obstruction (UUO) animal model. However, its role and mechanism in podocyte injury during diabetic nephropathy (DN) remains unclear. Diabetic mice model was established by intraperitoneal injection of streptozotocin (STZ) and oral administration of AEP inhibitor was adopted to inhibit AEP activity, and also, AEP knockout mice were employed. In vitro study, human podocyte cell line was employed, and subjected to different treatments after differentiation. RNA interfere and adenovirus expression vector were utilized to knockdown AEP or overexpression of AEP and cofilin 1 fragments respectively. Moreover, AEP recombinase was used to detect its substrate---cofilin 1 cleavage activity in vitro. In the present study, AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggests a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.

Glycosylation of FGFR4 in cholangiocarcinoma regulates receptor processing and cancer signaling.

Recent advances in targeted treatment for cholangiocarcinoma have focused on fibroblast growth factor (FGF) signaling. There are four receptor tyrosine kinases that respond to FGFs, and posttranslational processing has been demonstrated for each FGF receptor. Here, we investigated the role of N-linked glycosylation on the processing and function of FGFR4. We altered glycosylation through enzymatic deglycosylation, small molecule inhibition of glycosyltransferases, or through site-directed mutagenesis of selected asparagine residues in FGFR4. Signaling was tested through caspase activation, migration, and subcellular localization of FGFR4. Our data demonstrate that FGFR4 has multiple glycoforms, with predominant bands relating to the full-length receptor that has a high mannose- or hybrid-type form and a complex-type glycan form. We further identified a set of faster migrating FGFR4 bands that correspond to the intracellular kinase domain, termed FGFR4 intracellular domain (R4-ICD). These glycoforms and R4-ICD were detected in human cholangiocarcinoma tumor samples, where R4-ICD was predominant. Removal of glycans in intact cells by enzymatic deglycosylation resulted in increased processing to R4-ICD. Inhibition of glycosylation using NGI-1, an oligosaccharyltransferase inhibitor, reduced both high mannose- or hybrid- and complex-type glycan forms of FGFR4, increased processing and sensitized to apoptosis. Mutation of Asn-112, Asn-258, Asn-290, or Asn-311 to glutamine modestly reduced apoptosis resistance, while mutation of Asn-322 or simultaneous mutation of the other four asparagine residues caused a loss of cytoprotection by FGFR4. None of the glycomutants altered the migration of cancer cells. Finally, mutation of Asn-112 caused a partial localization of FGFR4 to the Golgi. Overall, preventing glycosylation at individual residues reduced the cell survival function of FGFR4 and receptor glycosylation may regulate access to an extracellular protease or proteolytic susceptibility of FGFR4.

Computational analysis of the mechanism of nonenzymatic peptide bond cleavage at the C-terminal side of an asparagine residue

Computational analysis of the mechanism of nonenzymatic peptide bond cleavage at the C-terminal side of an asparagine residue

Ever-expanding NGLY1 biology.

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in humans) is a deglycosylating enzyme that is widely conserved in eukaryotes. This enzyme is involved in the degradation of misfolded N-glycoproteins that are destined for proteasomal degradation in the cytosol, a process that is called endoplasmic reticulum-associated degradation. Although the physiological significance of NGLY1 remained unknown until recently, the discovery of NGLY1 deficiency, a human genetic disorder bearing mutations in the NGLY1 gene, has led to explosive research progress regarding the functional characterization of this enzyme. For example, it is now known that NGLY1 can also act as an 'editing enzyme' to convert N-glycosylated asparagine residues to aspartate residues, thus introducing negative charges into a core peptide and modulating the function of the target molecule. Diverse biological processes have also been found to be affected by compromised NGLY1 activity. In this special issue, recent research progress on the functional characterization of NGLY1 and its orthologues in worm/fly/rodents, assay methods/biomarkers useful for the development of therapeutics and the comprehensive transcriptome/proteome of NGLY1-KO cells as well as patient-derived cells are discussed.

Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling

Significance The uptake of molecules into cells, known as endocytosis, requires membrane invagination and the formation of vesicles. A version of endocytosis that is independent of actin polymerization is aided by the assembly of membraneless biomolecular condensates at the site of membrane invagination. Here, we show that endocytic condensates are viscoelastic bodies that concentrate key proteins with prion-like domains to enable membrane remodeling. A distinct molecular grammar, namely the preference for glutamine versus asparagine residues, underlies the cohesive interactions that give rise to endocytic condensates. We incorporate material properties inferred using active rheology into a mechanical model to explain how cohesive interactions within condensates and interfacial tensions among condensates, membranes, and the cytosol can drive membrane invagination to initiate endocyosis.

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing asparagine

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

Top-down proteomic identification of plasmid and host proteins produced by pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry.

Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.

Network-based identification of miRNAs and transcription factors and in silico drug screening targeting δ-secretase involved in Alzheimer's disease

BackgroundSystem medicine approaches have played a pivotal role in identifying novel disease networks especially in miRNA research. It is no wonder that miRNAs are implicated in multiple clinical conditions, allowing us to establish the hubs and nodes for network models of Alzheimer's Disease (AD). AD is an age-related, progressive, irreversible, and multifactorial neurodegenerative disorder characterized by cognitive and memory impairment and is the most common cause of dementia in older adults. Worldwide, around 50 million people have dementia, and there are nearly 10 million new cases every year. δ-secretase, also known as asparagine endopeptidase (AEP) or legumain (LGMN), is a lysosomal cysteine protease that cleaves peptide bonds C-terminally to asparagine residues in both amyloid precursor protein (APP) and tau, mediating the amyloid-β and tau pathology in AD. The patient's miRNA expression was found to be deregulated in the brain, extracellular fluid, blood plasma, and serum. MethodsProtein-Protein Interaction (PPI) networks of LGMN or δ-secretase were constructed using the Genemania database. Network Analyzer, a Cytoscape plugin, analyzed the network topological properties of LGMN. miRNAs related to Alzheimer's were extracted from the HMDD (Human microRNA Disease Database) and experimentally verified miRNA-gene interaction was obtained by searching miRWalk. Starbase v2.0 and miRanda were used for screening miRNA of LGMN genes. Moreover, to understand the regulatory mechanism in AD, we have screened major transcription factors of LGMN targeted genes using the Network Analyst 3.0, TRRUST (v2.0) server, and ENCODE. The Genotype-Tissue Expression (GTEx) and BEST tool were used to investigate the expression pattern of the LGMN gene. In parallel, we performed in-silico drug designing of the novel inhibitor scaffold of δ-secretase as powerful therapeutic targets by using the concept of scaffolds and frameworks. In this context, this study also aimed at identifying effective small molecule inhibitors targeting δ-secretase. ResultsAmong the 16 experimentally verified miRNAs, Network analysis of the LGMN and its associated miRNA identify novel hsa-miRNA-106a-5p and hsa-miRNA-34a-5p being more expressed in the brain. Our in silico high throughput screening, followed by XP docking revealed Oprea1 as the lead. Molecular dynamic simulations of the δ-secretase-docked complex have been carried out for a time period of 200 ns and revealed that Root Mean Square Deviation (RMSD) of the protein Cα-backbone with respect to its starting position increased to 1.20 Å for the first 25 ns of the trajectory and then became stable around 0.6 Å in the last 170 ns course of the simulation. The radius of gyration (RGYR) reveals that compactness was maintained till the end of simulations. ConclusionNetwork analysis of LGMN associated miRNAs lead to the identification of two novel miRNAs, being highly expressed in the brain. This study also lead to the identification and expression of 10 Transcription factors associated with LGMN. Expression Heatmap results show high and continuous expression of LGMN in most of the regions of the brain, especially in the frontal cortex. Further, in silico drug analysis led us to the identification of Oprea1 which could be taken for further investigation to explore its potential for AD therapy.

Synthesis of PNGase-resistant N-glycopeptide containing an α-anomeric glycosidic linkage

N-glycans in eukaryotic proteins are commonly attached to asparagine residues via β-N-glycosidic linkages, which are susceptible to glycosidases, such as PNGase. Here we report the preparation of α-N-glycosylated peptides based on the solid-phase peptide synthesis strategy using an α-N-GlcNAc-containing asparagine building block, and a transglycosylation reaction of oligosaccharide oxazoline promoted by endoglycosidases CCN180H. The resultant glycopeptide, bearing the complex-type α-N-sialyl undecasaccharide, exhibits resistance against PNGase F, which may be of potential use in studying the catabolism of N-glycans and glycoengineering those peptide-based therapeutics.

A novel insertion mutation in atlastin 1 is associated with spastic quadriplegia, increased membrane tethering, and aberrant conformational switching

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neuropathies affecting upper motor neurons and causing progressive gait disorder. Mutations in the gene SPG3A/atlastin-1 (ATL1), encoding a dynamin superfamily member, which utilizes the energy from GTP hydrolysis for membrane tethering and fusion to promote the formation of a highly branched, smooth endoplasmic reticulum (ER), account for approximately 10% of all HSP cases. The continued discovery and characterization of novel disease mutations are crucial for our understanding of HSP pathogenesis and potential treatments. Here, we report a novel disease-causing, in-frame insertion in the ATL1 gene, leading to inclusion of an additional asparagine residue at position 417 (N417ins). This mutation correlates with complex, early-onset spastic quadriplegia affecting all four extremities, generalized dystonia, and a thinning of the corpus callosum. We show using limited proteolysis and FRET-based studies that this novel insertion affects a region in the protein central to intramolecular interactions and GTPase-driven conformational change, and that this insertion mutation is associated with an aberrant prehydrolysis state. While GTPase activity remains unaffected by the insertion, membrane tethering is increased, indicative of a gain-of-function disease mechanism uncommon for ATL1-associated pathologies. In conclusion, our results identify a novel insertion mutation with altered membrane tethering activity that is associated with spastic quadriplegia, potentially uncovering a broad spectrum of molecular mechanisms that may affect neuronal function.

Antigen Glycosylation Is a Central Regulator of CAR T Cell Efficacy

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) have revolutionized the management of relapsed and refractory B cell malignancies. Despite high initial response rates, many patients with acute lymphoblastic leukemia (ALL) ultimately relapse after CART19. In contrast, most patients with non-Hodgkin lymphoma experience only partial or no responses. Collectively, <50% of patients treated with CART19 achieve durable disease remission. Identification of the biology responsible for these failures is central to improving CAR T cell efficacy. Several clinical reports have demonstrated that a common cause of resistance to CART19 is antigen escape, in which ALL clones emerge that have lost surface expression of CD19. The mechanisms of antigen escape that have been recognized to date all rely on disruption of CD19 genomic loci or transcribed CD19 mRNA; alterations of fully-translated CD19 protein that lead to CART19 failure have not been described.To identify pathways responsible for enabling tumor-intrinsic resistance to CART19 we performed a genome-wide loss-of-function screen in the Nalm6 ALL cell line. The second-most enriched gene in this screen was SPPL3 (Figure 1a), encoding a Golgi-resident aspartyl protease. Previous studies have determined that SPPL3 functions to broadly limit protein glycosylation by cleaving glycosyltransferases from the Golgi membrane, impairing their ability to add complex glycans to proteins as they pass through the Golgi (Voss M. et al. EMBO, 2014). Using targeted genomic disruption, we confirmed that loss of SPPL3 results in resistance to CART19 in human ALL and non-Hodgkin lymphoma models (Figures 1b-c). CART19 cells exposed to SPPL3KO ALL demonstrated significantly lower expression of CD69, PD1, Tim3 and CD107a, as well as less activation of the central T cell transcription factors NFAT and NFκB, indicating a global suppression of T cell stimulation.Consistent with its known function, loss of SPPL3 resulted in increased addition of complex glycans to CD19. Surface staining of SPPL3KO cells revealed that CD19 antibodies were less capable of binding this hyperglycosylated CD19. This included decreased binding of the antibody used to construct the anti-CD19 CAR (clone FMC63). Protein modeling revealed that an asparagine residue known to be normally glycosylated on CD19 (N125) is in close physical proximity to the FMC63 binding site (Figure 1d), suggesting that the addition of complex glycans at this site may be responsible for disruption of CAR binding that led to impaired T cell activation. We next turned our attention to CD22, another B cell antigen that is normally glycosylated and the target of CAR therapy. In contrast to CD19, loss of SPPL3 had no impact on CD22 glycosylation or antibody binding. Similarly, loss of SPPL3 did not enable resistance to CD22-targeted CAR T cells. These findings substantiated our hypothesis loss of SPPL3 lead to CART19 failure directly via modifying CD19 glycosylation, and not through another CD19-independent mechanism. To further validate the impact of CD19 glycosylation in regulating CART19 efficacy, we over-expressed SPPL3 in ALL cells, previously shown to promote global hypoglycosylation. We confirmed decreased glycosylation of CD19 (Figure 1e), and found that this resulted in loss of FMC63 binding to CD19 and complete resistance to CART19 activity (Figure 1f).In summary, our findings identify that changes to CD19 glycosylation, either enhanced or decreased, impair the ability of CARs to bind and initiate T cell effector function against malignant B cells. Further, these data identify post-translational protein modification as a novel mechanism of antigen escape from CAR-based T cell immunotherapy. [Display omitted] DisclosuresRuella: AbClon: Consultancy, Research Funding; viTToria biotherapeutics: Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; Tmunity: Patents & Royalties. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Effects of Normal and Aberrant Glycosylation on the Stability of α1-Anti Trypsin Through Molecular Dynamic Simulation

The Alpha-1 antitrypsin belonging to serpin family is a protease inhibitor, the level of which rises by a factor of ten during inflammation Purpose: To investigate the stability of normal and aberrantly glycosylated α1-antitrypsin through molecular dynamics simulation Study Design: Experimental study Methodology: Current project was conducted in the department of Biochemistry at Institute of Basic Medical Sciences Khyber Medical University, Peshawar. A1AT FASTA sequence was retrieved from UniProt database (UniProt ID: P01009). Post-Translational Modifications (PTM) regions were identified from the same database. GLYCONNECT database was used to understand N-linked glycation with the asparagine residues found at position 70, 107, and 271 amino acid residue regions Statistical analysis: Different bioinformatics analyses such that Root Mean Square Deviation, Radius of gyration, Root Mean Square fluctuation, Hydrogen-bonding, Secondary Structure Determination, and Principal Component Analysis were executed for 100 ns molecular dynamics simulation run Results: RMSd, RMSf, and Rgyr significantly differ between the native type and cancer isoform. More H-bonding and strong protein stability and folding were seen in the native type. PCA analysis further confirms native type compact motion in the parallel direction during MD simulation Conclusion: It was concluded that glycated protein appears to have high structural stability than its aberrant glycated protein. However, it will be utilized for the prompt production of the anti-cancer drugs to effectively treating cancer disease Key Words: Alpha-1 Antitrypsin, Aberrant Glycosylation and Molecular Dynamic Simulation.

Kinetic Studies of the Hydrogen Atom Transfer in a Hypoxia-Sensing Enzyme, FIH-1: KIE and O2 Reactivity.

Cellular hypoxia plays a crucial role in tissue development and adaptation to pO2. Central to cellular oxygen sensing is factor-inhibiting HIF-1α (FIH), an α-ketoglutarate (αKG)/non-heme iron(II)-dependent dioxygenase that hydroxylates a specific asparagine residue of hypoxia inducible factor-1α (HIF-1α). The high KM(O2) and rate-limiting decarboxylation step upon O2 activation are key features of the enzyme that classify it as an oxygen sensor and set it apart from other αKG/Fe(II)-dependent dioxygenases. Although the chemical intermediates following decarboxylation are presumed to follow the consensus mechanism of other αKG/Fe(II)-dependent dioxygenases, experiments have not previously demonstrated these canonical steps in FIH. In this work, a deuterated peptide substrate was used as a mechanistic probe for the canonical hydrogen atom transfer (HAT). Our data show a large kinetic isotope effect (KIE) in steady-state kinetics (Dkcat = 10 ± 1), revealing that the HAT occurs and is partially rate limiting on kcat. Kinetic studies showed that the deuterated peptide led FIH to uncouple O2 activation and provided the opportunity to spectroscopically observe the ferryl intermediate. This enzyme uncoupling was used as an internal competition with respect to the fate of the ferryl intermediate, demonstrating a large observed KIE on the uncoupling (Dk5 = 1.147 ± 0.005) and an intrinsic KIE on the HAT step (Dk > 15). The close energy barrier between αKG decarboxylation and HAT distinguishes FIH as an O2-sensing enzyme and is crucial for ensuring substrate specificity in the regulation of cellular O2 homeostasis.

Computational Analysis of the Mechanism of Nonenzymatic Peptide Bond Cleavage at the C-Terminal Side of an Asparagine Residue.

The nonenzymatic peptide bond cleavage at the C-terminal side of Asn residues is a protein post-translational modification that occurs under physiological conditions. This reaction proceeds much slower than the deamidation of the Asn side chain and causes denaturation and hypofunction of proteins. The peptide bond cleavage of Asn is detected primarily in crystallins and aquaporin 0 in the eye lens. Therefore, cleavage is thought to be involved in age-related cataracts. In this study, to clarify the mechanism underlying succinimide formation for the peptide bond cleavage of the Asn residue, we performed quantum chemical calculations on the model compound Ace-Asn-Gly-Nme (Ace = acetyl and Nme = methylamino). The density functional theory with the B3LYP/6-31+G(d,p) level of theory was used to obtain optimized geometries. The results suggested that the reaction proceeds through two steps, cyclization and C-terminal fragment release, and the required proton transfers can be mediated by H2PO4– and HCO3– ions. The conformational change of the main chain on the N-terminal side of Asn was needed for the C-terminal fragmentation step, and a separate conformational change at the C-terminal side was required for the cyclization step. Furthermore, the calculated activation barriers of the reactions catalyzed by the H2PO4– ion (130 kJ mol–1) and the HCO3– ion (123 kJ mol–1) were sufficiently low for the reactions to occur under normal physiological conditions.