Differences In Amino Acid Sequence Research Articles

Risk assessment of homologous variants of biotech trait proteins using a bridging approach

ABSTRACT A transgenic protein is frequently expressed as different homologous variants in genetically modified crops due to differential processing of targeting peptides or optimization of activity and specificity. The aim of this study was to develop a science-based approach for risk assessment of homologous protein variants using dicamba mono-oxygenase (DMO) as a case study. In this study, DMO expressed in the next-generation dicamba-tolerant maize, sugar beet and soybean crops exhibited up to 27 amino acid sequence differences in the N-terminus. Structure modeling using AlphaFold, ESMFold and OpenFold demonstrates that these small N-terminal extensions lack an ordered secondary structure and do not disrupt the DMO functional structure. Three DMO variants were demonstrated to have equivalent immunoreactivity and functional activity ranging from 214 to 331 nmol/min/mg. Repeated toxicity studies using each DMO variant found no test substance-related adverse effects. These results support that homologous protein variants, which have demonstrated physicochemical and functional equivalence, can leverage existing safety data from one variant without requiring additional de novo safety assessments.

Comparative Analysis of Intestinal TLR4 Gene Expression and Functional Verification in Lepus yarkandensis and Oryctolagus cuniculus.

Lepus yarkandensis live year-round in harsh desert environments and are less susceptible to enteritis. The living conditions of Oryctolagus cuniculus in captivity were suitable, but they were highly susceptible to death by Gram-negative bacteria infected with inflammatory bowel disease complex.TLR4 is closely related to the occurrence of enteritis, and the neighbor-joining topology based on the 12S rDNA sequences showed that the relationship between O. cuniculus and L. yarkandensis is as high as 98%.Therefore, we chose O. cuniculus and L. yarkandensis for comparative study.The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) in the regulation of immunity and inflammation in the intestinal tract of L. yarkandensis. In this study, the TLR4 gene was cloned for the first time in the colon of L. yarkandensis. The expression of TLR4 in the intestinal tissues of L. yarkandensis and O. cuniculus was detected by histological observation, real-time fluorescence quantification PCR(qRT-PCR), and protein blotting (Western blot).An LPS-induced cell inflammation model was constructed in vitro, and ELISA was used to examine the effect of pEGFP-N1-TLR4 and siRNA knockout on the anti-inflammatory ability of the TLR4 gene. The results showed that the open reading frame of the L. yarkandensis TLR4 gene was 2520bp in length. Compared with the sequence of O. cuniculus, there were 15 differences in the TLR4 amino acid sequence of L. yarkandensis, 12 of which occurred in the LRR domain and 2 in the TIR domain, and the sequence changed from G to D at position 298. Immunohistochemistry showed that TLR4 was mainly expressed in the epithelial cells of the colon L. yarkandensis, and the expression level of TLR4 in the cecum and colon was significantly lower compared with that of O. cuniculus. qRT-PCR and Western blot results showed that the expression level of TLR4 in the colon of L. yarkandensis was significantly lower than that of O. cuniculus. At the cellular level, ELISA showed that overexpression of the TLR4 protein in L. yarkandensis could reduce the LPS-induced inflammatory response. Therefore, according to the above results, the protein structure and function of L. yarkandensis TLR4 may be different due to the change of nucleotide, which affects its binding with LPS and the activation of downstream molecules, so that L. yarkandensis is not prone to enteritis and can adapt to the harsh desert environment for a long time. This study also laid the foundation for improving the disease resistance of O. cuniculus and promoting the development and utilization of genes in L. yarkandensis.

Broadly cross-reactive immune responses in chickens immunized with chimeric virus-like particles of nodavirus displaying the M2e originated from avian and human influenza A viruses

Avian influenza A viruses (IAVs) pose a persistent threat to poultry industry worldwide, despite the presence of vaccines. Additionally, reverse-zoonosis transmission potentially introduces human-originated IAVs into poultry and complicates the efforts to control the spread of influenza. Current avian influenza vaccines are primarily based upon the rapidly mutating hemagglutinin (HA) and neuraminidase (NA) glycoproteins, which limit their efficacy against diverse strains of IAVs. Hence, the highly conserved ectodomains of matrix 2 protein (M2e) of IAVs are widely studied as alternatives to the HA and NA. However, the differences in the M2e amino acid sequences between avian and human IAVs generate antibodies that do not cross-react reciprocally with IAVs from other origins. To broaden and enhance the immunogenicity of M2e, we fused two copies each of the M2e derived from avian and human IAVs at the C-terminal end of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (NvC). Transmission electron microscopic and dynamic light scattering analyses revealed that the chimeric protein self-assembled into virus-like particles (VLPs). Immunization of chickens with the chimeric VLPs demonstrated a robust induction of broadly reactive immune responses against both the M2e of avian and human IAVs. Additionally, the chimeric VLPs elicited the production of cytotoxic T lymphocytes (CTL), macrophages, as well as a well-balanced Th1 and Th2 population, indicating their potential in activating cell-mediated immune responses in chickens. Furthermore, the chimeric VLPs triggered the production of both Th1- and Th2-cytokines, attesting their potential in mounting a robust and balanced immune response in avian species. This study demonstrated the potential of these chimeric VLPs in stimulating and broadening cross-reactive immune responses in chickens against both avian and human IAVs.

Genome-Wide Identification, Expression Analysis and Functional Study of DELLA Genes in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

DELLA protein is a crucial factor which played pivotal roles in regulating numerous intriguing biological processes in plant development and abiotic stress responses. However, little is known about the function and information of DELLA protein in Chinese cabbage. Using 5 DELLA gene sequences in Arabidopsis Thaliana as probes, 5 DELLA genes in Chinese cabbage were identified by Blast search in Chinese cabbage database (Brassica database (BRAD)). The National Center for Biotechnology Information (NCBI), ExPaSy, SWISS-MODEL, DNAMAN, MEGA 11, PlantCARE were used to identify and analyze the DELLA gene family of Chinese cabbage. Gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The function of BraA10gRGL3 was verified by overexpression and phenotypic analysis of BraA10gRGL3 and yeast hybrid. In this study, 5 BraDELLAs homologous to Arabidopsis thaliana were identified and cloned based on the Brassica database, namely, BraA02gRGL1, BraA05gRGL2, BraA10gRGL3, BraA06gRGA and BraA09gRGA. All BraDELLAs contain the DELLA, TVHYNP, and GRAS conserved domains. Cis-element analysis revealed that the promoter regions of these 5 DELLA genes all contain light-responsive elements, TCT motif, I-box, G-box, and box 4, which are associated with GA signaling. Transcriptome analysis results proved that the expression of BraA02gRGL1, BraA05gRGL2, and BraA10gRGL3 in Y2 at different growth stages were lower than them in Y7, which is consistent with the phenotype that Y7 exhibited stronger stress tolerance than Y2. It is worth emphasizing that even through the overexpression of BraA10gRGL3-Y7 in Arabidopsis resulted in smaller leaf size and lower fresh weight compared to the wild type (WT) Arabidopsis: Columbia, a stronger response to abiotic stresses was observed in BraA10gRGL3-Y7. It indicated that BraA10gRGL3-Y7 can improve the stress resistance of plants by inhibiting their growth. Moreover, the yeast two-hybrid experiment confirmed that BraA10gRGL3-Y7 can interacted with BraA05gGID1a-Y7, BraA04gGID1b1, BraA09gGID1b3-Y2, and BraA06gGID1c, whereas BraA10gRGL3-Y2 cannot interact with any BraGID1. Collectively, BraDELLAs play important role in plant development and response to abiotic stress. The differences in amino acid sequences between BraA10gRGL3-Y2 and BraA10gRGL3-Y7 may result in variations in their protein binding sites, thus affecting their interaction with the BraGID1 family proteins. This systematic analysis lays the foundation for further study of the functional characteristics of DELLA genes of Chinese cabbage.

Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

Urotensin II (UII) and UII-related peptide (URP) and their receptor, UT, comprise the UII system. Although UII is conserved across all vertebrates and shares a cyclic hexapeptide core-sequence motif of CFWKYC, there are differences in N-terminal amino acid sequences between species. The kidney is a significant source of UII, the level of which is amplified in infants with chronic kidney disease. We have previously shown that human UII increases renal vascular resistance (RVR) and reduces renal blood flow in neonatal pigs. However, the effect of pig UII (pUII) on renal microcirculation remains unclear. Since the mechanisms of renal ischemia-reperfusion (IR) injury include kidney hypoperfusion, increased activity of the UII system may contribute to ischemic acute kidney injury (AKI). Here, we demonstrate that kidney tissue UII, URP, and UT and renal vascular UT expression levels are higher in neonatal pigs than in adults. Intrarenal artery infusion of pUII reduced kidney perfusion and increased RVR, which was reversed by urantide, a peptide UT inhibitor. One hour of renal ischemia, followed by 8 hours of reperfusion, did not alter kidney tissue UT but upregulated kidney tissue UII and URP and renal vascular UT expression levels. Urantide mitigated renal IR-induced increase in AKI biomarkers. Urantide also attenuated renal IR-induced increases in serum interleukin-1beta, interleukin-10, interferon-α, and morphological kidney injury. The enzyme that converts prepro UII to active UII has been proposed to be serine protease furin. Kidney tissue expression of furin is higher in neonatal pigs compared with adults. Renal IR also upregulated furin expression in neonatal pig kidneys. Exposure of primary neonatal pig renal epithelial cells to the cellular model of IR (cIR) increased furin levels. cIR also increased UII production in the cells, an effect attenuated by furin inhibitor SS3. These findings suggest that in pigs, 1) the UII system is upregulated in immature kidneys, 2) renal IR upregulates furin, UII, and URP in kidney tissues and UT in renal blood vessels of neonates, 3) ischemic activation of furin promotes UII biosynthesis by renal epithelial cells, and 4) pharmacological inhibition of UT mitigates renal IR-induced AKI. We conclude that the UII systems may contribute to IR-induced neonatal renal insuffciency. NIH R01DK120595 R01DK127625. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A systemic approach to identifying sequence frameworks that decrease mAb production in a transient Chinese hamster ovary cell expression system.

Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their "developability" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these "difficult-to-express" (DTE) mAbs has shown that these phenotypes are driven by post-translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG1κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines.

Evolved changes in phenotype across skeletal muscles in deer mice native to high altitude.

The cold and hypoxic conditions at high altitude necessitate high metabolic O2 demands to support thermogenesis while hypoxia reduces O2 availability. Skeletal muscles play key roles in thermogenesis, but our appreciation of muscle plasticity and adaptation at high altitude has been hindered by past emphasis on only a small number of muscles. We examined this issue in deer mice (Peromyscus maniculatus). Mice derived from both high-altitude and low-altitude populations were born and raised in captivity and then acclimated as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 wk). Maximal activities of citrate synthase (CS), cytochrome c oxidase (COX), β-hydroxyacyl-CoA dehydrogenase (HOAD), hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) were measured in 20 muscles involved in shivering, locomotion, body posture, ventilation, and mastication. Principal components analysis revealed an overall difference in muscle phenotype between populations but no effect of hypoxia acclimation. High-altitude mice had greater activities of mitochondrial enzymes and/or lower activities of PK or LDH across many (but not all) respiratory, limb, core and mastication muscles compared with low-altitude mice. In contrast, chronic hypoxia had very few effects across muscles. Further examination of CS in the gastrocnemius showed that population differences in enzyme activity stemmed from differences in protein abundance and mRNA expression but not from population differences in CS amino acid sequence. Overall, our results suggest that evolved increases in oxidative capacity across many skeletal muscles, at least partially driven by differences in transcriptional regulation, may contribute to high-altitude adaptation in deer mice.NEW & NOTEWORTHY Most previous studies of muscle plasticity and adaptation in high-altitude environments have focused on a very limited number of skeletal muscles. Comparing high-altitude versus low-altitude populations of deer mice, we show that a large number of muscles involved in shivering, locomotion, body posture, ventilation, and mastication exhibit greater mitochondrial enzyme activities in the high-altitude population. Therefore, evolved increases in mitochondrial oxidative capacity across skeletal muscles contribute to high-altitude adaptation.

The spike protein of the apathogenic Beaudette strain of avian coronavirus can elicit a protective immune response against a virulent M41 challenge.

The avian Gammacoronavirus infectious bronchitis virus (IBV) causes major economic losses in the poultry industry as the aetiological agent of infectious bronchitis, a highly contagious respiratory disease in chickens. IBV causes major economic losses to poultry industries across the globe and is a concern for global food security. IBV vaccines are currently produced by serial passage, typically 80 to 100 times in chicken embryonated eggs (CEE) to achieve attenuation by unknown molecular mechanisms. Vaccines produced in this manner present a risk of reversion as often few consensus level changes are acquired. The process of serial passage is cumbersome, time consuming, solely dependent on the supply of CEE and does not allow for rapid vaccine development in response to newly emerging IBV strains. Both alternative rational attenuation and cell culture-based propagation methods would therefore be highly beneficial. The majority of IBV strains are however unable to be propagated in cell culture proving a significant barrier to the development of cell-based vaccines. In this study we demonstrate the incorporation of a heterologous Spike (S) gene derived from the apathogenic Beaudette strain of IBV into a pathogenic M41 genomic backbone generated a recombinant IBV denoted M41K-Beau(S) that exhibits Beaudette's unique ability to replicate in Vero cells, a cell line licenced for vaccine production. The rIBV M41K-Beau(S) additionally exhibited an attenuated in vivo phenotype which was not the consequence of the presence of a large heterologous gene demonstrating that the Beaudette S not only offers a method for virus propagation in cell culture but also a mechanism for rational attenuation. Although historical research suggested that Beaudette, and by extension the Beaudette S protein was poorly immunogenic, vaccination of chickens with M41K-Beau(S) induced a complete cross protective immune response in terms of clinical disease and tracheal ciliary activity against challenge with a virulent IBV, M41-CK, belonging to the same serogroup as Beaudette. This implies that the amino acid sequence differences between the Beaudette and M41 S proteins have not distorted important protective epitopes. The Beaudette S protein therefore offers a significant avenue for vaccine development, with the advantage of a propagation platform less reliant on CEE.

Adrenodoxin allosterically alters human cytochrome P450 11B enzymes to accelerate substrate binding and decelerate release.

Two human mitochondrial membrane CYP11B enzymes play a pivotal role in steroidogenesis. CYP11B1 generates the major glucocorticoid cortisol, while CYP11B2 catalysis yields the primary mineralocorticoid aldosterone. Catalysis by both requires electron delivery by a soluble iron-sulfur adrenodoxin redox partner. However recent studies have shown that adrenodoxin/CYP11B interaction alone allosterically increases substrate and inhibitor affinity as exhibited by decreased dissociation constant (K d) values. The current study moves beyond such equilibrium studies, by defining adrenodoxin effects on the rates of P450 ligand binding and release separately. Stopped-flow data clearly demonstrate that adrenodoxin interaction with the P450 proximal surfaces increases ligand binding in both P450 CYP11B active sites by increasing the on rate constant and decreasing the off rate constant. As substrate entry and exit from the sequestered P450 active site requires conformational changes on the distal side of the P450 enzyme, a likely explanation is that adrenodoxin binding allosterically modulates CYP11B conformational changes. The 93% identical CYP11B enzymes can bind and hydroxylate each other's native substrates differing only by a hydroxyl. However, CYP11B1 exhibits monophasic substrate binding and CYP11B2 biphasic substrate binding, even when the substrates are swapped. This indicates that small differences in amino acid sequence between human CYP11B1 and CYP11B2 enzymes are more functionally important in ligand binding and could suggest avenues for more selective inhibition of these drug targets. Both protein/protein interactions and protein/substrate interactions are most likely to act by modulating CYP11B conformational dynamics.

Production and characterization of homologous protoporphyrinogen IX oxidase (PPO) proteins: Evidence that small N-terminal amino acid changes do not impact protein function.

Transgenic soybean, cotton, and maize tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides have been developed by introduction of a bacterial-derived PPO targeted into the chloroplast. PPO is a membrane-associated protein with an intrinsic tendency for aggregation, making expression, purification, and formulation at high concentrations difficult. In this study, transgenic PPO expressed in three crops was demonstrated to exhibit up to a 13 amino acid sequence difference in the N-terminus due to differential processing of the chloroplast transit peptide (CTP). Five PPO protein variants were produced in and purified from E. coli, each displaying equivalent immunoreactivity and functional activity, with values ranging from 193 to 266 nmol min-1 mg-1. Inclusion of an N-terminal 6xHis-tag or differential processing of the CTP peptide does not impact PPO functional activity. Additionally, structural modeling by Alphafold, ESMfold, and Openfold indicates that these short N-terminal extensions are disordered and predicted to not interfere with the mature PPO structure. These results support the view that safety studies on PPO from various crops can be performed from a single representative variant. Herein, we report a novel and robust method for large-scale production of PPO, enabling rapid production of more than 200 g of highly active PPO protein at 99% purity and low endotoxin contamination. We also present a formulation that allows for concentration of active PPO to > 75 mg/mL in a buffer suitable for mammalian toxicity studies.

Undesirable protein sequence variations in maize genes that confer resistance to fungal pathogens and insect pests

Diseases and insect pests greatly impact sustainable production in maize. Maize inbred lines have varying levels of resistance to these pathogens and insects, but little is known about the diversity of their resistance proteins. In this study, genes encoding seven proteins that are involved in resistance to insects and pathogens in maize were analyzed in 46 maize inbred lines to elucidate the differences in amino acid sequences. The proteins of interest are superlectin, maizewin, hydrolase, geranyl geranyl transferase, quinone oxidoreductase, AIL1, and defensin. The protein sequences encoded by genes for superlectin, AIL1, and defensin were found to be disrupted in some maize inbreds but were conserved in others. The characterized disruptions resulted from single amino acid changes, insertions, or deletions. While the effect of single amino acid changes is hard to predict, insertions and deletions likely disrupt protein function, increasing the susceptibility of maize plants to insects and/or diseases. Functional resistance genes can be incorporated from the identified maize inbreds into commercial hybrids to promote enhanced insect and pathogen resistance.

The Photoprotective Protein PsbS from Green Microalga Lobosphaera incisa: The Amino Acid Sequence, 3D Structure and Probable pH-Sensitive Residues.

PsbS is one of the key photoprotective proteins, ensuring the tolerance of the photosynthetic apparatus (PSA) of a plant to abrupt changes in irradiance. Being a component of photosystem II, it provides the formation of quenching centers for excited states of chlorophyll in the photosynthetic antenna with an excess of light energy. The signal for "turning on" the photoprotective function of the protein is an excessive decrease in pH in the thylakoid lumen occurring when all the absorbed light energy (stored in the form of transmembrane proton potential) cannot be used for carbon assimilation. Hence, lumen-exposed protonatable amino acid residues that could serve as pH sensors are the essential components of PsbS-dependent photoprotection, and their pKa values are necessary to describe it. Previously, calculations of the lumen-exposed protonatable residue pKa values in PsbS from spinach were described in the literature. However, it has recently become clear that PsbS, although typical of higher plants and charophytes, can also provide photoprotection in green algae. Namely, the stress-induced expression of PsbS was recently shown for two green microalgae species: Chlamydomonas reinhardtii and Lobosphaera incisa. Therefore, we determined the amino acid sequence and modeled the three-dimensional structure of the PsbS from L. incisa, as well as calculated the pKa values of its lumen-exposed protonatable residues. Despite significant differences in amino acid sequence, proteins from L. incisa and Spinacia oleracea have similar three-dimensional structures. Along with the other differences, one of the two pH-sensing glutamates in PsbS from S. oleracea (namely, Glu-173) has no analogue in L. incisa protein. Moreover, there are only four glutamate residues in the lumenal region of the L. incisa protein, while there are eight glutamates in S. oleracea. However, our calculations show that, despite the relative deficiency in protonatable residues, at least two residues of L. incisa PsbS can be considered probable pH sensors: Glu-87 and Lys-196.

Mitogenome of the stink bug Aelia fieberi (Hemiptera: Pentatomidae) and a comparative genomic analysis between phytophagous and predatory members of Pentatomidae.

Aelia fieberi Scott, 1874 is a pest of crops. The mitogenome of A. fieberi (OL631608) was decoded by next-generation sequencing. The mitogenome, with 41.89% A, 31.70% T, 15.44% C and 10.97% G, is 15,471 bp in size. The phylogenetic tree showed that Asopinae and Phyllocephalinae were monophyletic; however, Pentatominae and Podopinae were not monophyletic, suggesting that the phylogenetic relationships of Pentatomoidae are complex and need revaluation and revision. Phytophagous bugs had a ~20-nucleotide longer in nad2 than predatory bugs. There were differences in amino acid sequence at six sites between phytophagous bugs and predatory bugs. The codon usage analysis indicated that frequently used codons used either A or T at the third position of the codon. The analysis of amino acid usage showed that leucine, isoleucine, serine, methionine, and phenylalanine were the most abundant in 53 species of Pentatomoidae. Thirteen protein-coding genes were evolving under purifying selection, cox1, and atp8 had the strongest and weakest purifying selection stress, respectively. Phytophagous bugs and predatory bugs had different evolutionary rates for eight genes. The mitogenomic information of A. fieberi could fill the knowledge gap for this important crop pest. The differences between phytophagous bugs and predatory bugs deepen our understanding of the effect of feeding habit on mitogenome.

Strain-dependent role of copper in prion disease through binding to histidine residues in the N-terminal domain of prion protein.

The cellular prion protein, PrPC , is a copper-binding protein abundantly expressed in the brain, particularly by neurons, and its conformational conversion into the amyloidogenic isoform, PrPSc , plays a key pathogenic role in prion diseases. However, the role of copper binding to PrPC in prion diseases remains unclear. Here, we fed mice with a low-copper or regular diet and intracerebrally inoculated them with two different mouse-adapted RML scrapie and BSE prions. Mice with a low-copper diet developed disease significantly but only slightly later than those with a regular diet after inoculation with BSE prions, but not with RML prions, suggesting that copper could play a minor role in BSE prion pathogenesis, but not in RML prion pathogenesis. We then generated two lines of transgenic mice expressing mouse PrP with copper-binding histidine (His) residues in the N-terminal domain replaced with alanine residues, termed TgPrP(5H > A)-7342/Prnp0/0 and TgPrP(5H > A)-7524/Prnp0/0 mice, and similarly inoculated RML and BSE prions into them. Due to 2-fold higher expression of PrP(5H > A) than PrPC in wild-type (WT) mice, TgPrP(5H > A)-7524/Prnp0/0 mice were highly susceptible to these prions, compared to WT mice. However, TgPrP(5H > A)-7342/Prnp0/0 mice, which express PrP(5H > A) 1.2-fold as high as PrPC in WT mice, succumbed to disease slightly, but not significantly, later than WT mice after inoculation with RML prions, but significantly so after inoculation with BSE prions. Subsequent secondary inoculation experiments revealed that amino acid sequence differences between PrP(5H > A) and WT PrPSc created no prion transmission barrier to BSE prions. These results suggest that copper-binding His residues in PrPC are dispensable for RML prion pathogenesis but have a minor effect on BSE prion pathogenesis. Taken together, our current results suggest that copper could have a minor effect on prion pathogenesis in a strain-dependent manner through binding to His residues in the N-terminal domain of PrPC .

UDP-glucose pyrophosphorylase as a target for regulating carbon flux distribution and antioxidant capacity in Phaeodactylum tricornutum

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme for polysaccharide synthesis, and its role in plants and bacteria is well established; however, its functions in unicellular microalgae remain ill-defined. Here, we perform bioinformatics, subcellular localization as well as in vitro and in vivo analyses to elucidate the functions of two UGPs (UGP1 and UGP2) in the model microalga Phaeodactylum tricornutum. Despite differences in amino acid sequence, substrate specificity, and subcellular localization between UGP1 and UGP2, both enzymes can efficiently increase the production of chrysolaminarin (Chrl) or lipids by regulating carbon flux distribution without impairing growth and photosynthesis in transgenic strains. Productivity evaluation indicate that UGP1 play a bigger role in regulating Chrl and lipid production than UGP2. In addition, UGP1 enhance antioxidant capacity, whereas UGP2 is involved in sulfoquinovosyldiacylglycerol (SQDG) synthesis in P. tricornutum. Taken together, the present results suggest that ideal microalgal strains can be developed for the industrial production of Chrl or lipids and lay the foundation for the development of methods to improve oxidative stress tolerance in diatoms.

Cannabidiol sensitizes TRPV2 channels to activation by 2-APB.

The cation-permeable TRPV2 channel is important for cardiac and immune cell function. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique, we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40°C) heat. Using cryo-EM, we uncover a new small-molecule binding site in the pore domain of rTRPV2 in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels are also activated by 2-APB and CBD and share multiple conserved features with TRPV2, but we find that strong sensitization by CBD is only observed in TRPV3, while sensitization for TRPV1 is much weaker. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of rTRPV2 channels engages multiple channel regions, and that the difference in sensitization strength between rTRPV2 and rTRPV1 channels does not originate from amino acid sequence differences at the CBD binding site or the pore domain. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels - their resilience to activation.

Shotgun Proteomics of Co-Cultured Leukemic and Bone Marrow Stromal Cells from Different Species as a Preliminary Approach to Detect Intercellular Protein Transfer

Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.

The seeding barrier between human and Syrian hamster prion protein amyloid fibrils is determined by β2-α2 loop sequence elements

Transmissive spongiform encephalopathies (TSE) are a group of neurodegenerative diseases caused by infectious protein particles, known as prions. Prions are formed from cellular prion proteins (PrP) and can be transmitted between different mammalian species. Subsequently, the host's PrPs are then converted to prions, followed by the onset of TSE. Interspecies prion infectivity is governed by the amino acid sequence differences of PrPs and prions' inability to replicate in a host is termed a species barrier. Here, we investigated the amino acid sequence determinants of species barrier between recombinant human (rHuPrP) and hamster (rShaPrP) prion protein amyloid fibrils. We discovered that a unidirectional species barrier between rShaPrP and rHuPrP amyloid fibrils exists. This barrier stems from the difference of amino acid sequences in the conserved β2-α2 loop region. Our results revealed that individual amino acids in the β2-α2 loop region are critical for overcoming the barrier between human and hamster prion protein amyloid fibrils in vitro. Furthermore, the barrier was only possible to observe through aggregation kinetics, as the secondary structure rHuPrP fibrils was not affected by the cross-seeding. Overall, we demonstrated the mechanistic pathway behind this interspecies barrier phenomenon, which increases our understanding of prion-related disease development.

PS-BPB07-1: DEVELOPMENT OF ANTI-THROMBOTIC VACCINE AGAINST HUMAN S100A9

Background & Purposes: In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we have reported that a newly designed antiplatelet vaccine against S100A9 could prevent thrombosis in the middle cerebral artery in mice by inhibiting S100A9/CD36 signaling in platelets. However, the difference in amino acid sequences in epitopes between mice and humans might be problematic for clinical use. In the present study, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys. Methods: First, we screened several candidate epitopes in mice and the selected epitope in human S100A9, which is common in monkey S100A9, was conjugated to Keyhole limpet hemocyanin. A mixture of the vaccine and Alum adjuvant was subcutaneously injected into 4 cynomolgus monkeys, three times every two weeks. Antibody production against human S100A9 was examined by ELISA or western blotting. Anti-thrombotic effects in collagen-induced thrombosis were examined in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey using an automated microchip flow-chamber system. Bleeding time was evaluated in two monkeys. Results: Based on results of computed epitope prediction, we first assessed whether 2 to 11, 97–106, or 104–113 amino acids, where the amino acid sequence is approximately similar between humans and monkeys, could raise antibodies against S100A9 in mice. Compared with the vaccine for the 2–11 amino acids epitopes, 97–106 amino acids and 104–113 amino acids demonstrated higher antigenicity, indicating that epitopes in the C-terminus are stronger than those in the N-terminus. Based on this result, we selected 102 to 112 amino acids as the epitope for the human S100A9 vaccine. When the monkeys were vaccinated, antibody titers against human S100A9 were significantly increased. Western blotting analysis showed that the produced antibody recognized human S100A9. Although the addition of pre-vaccinated serum into fresh whole blood did not affect thrombus formation, post-vaccination serum inhibited collagen-induced thrombosis formation in two monkeys. Anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects by vaccination. Notably, prolongation of bleeding time was not observed in vaccinated monkeys. Conclusions: Anti-human S100A9 vaccine revealed anti-thrombotic effects in some monkeys without affecting bleeding time. Although further studies are necessary to increase the effects and warrant the safety aspects, this vaccine could clarify the use of an anti-thrombotic vaccine in clinical settings.

Genetic Variants of Adrenoceptors.

Adrenoceptors are class A G-protein-coupled receptors grouped into three families (α1-, α2-, and β-adrenoceptors), each one including three members. All nine corresponding adrenoceptor genes display genetic variation in their coding and adjacent non-coding genomic region. Coding variants, i.e., nucleotide exchanges within the transcribed and translated receptor sequence, may result in a difference in amino acid sequence thus altering receptor function and signaling. Such variants have been intensely studied in vitro in overexpression systems and addressed in candidate-gene studies for distinct clinical parameters. In recent years, large cohorts were analyzed in genome-wide association studies (GWAS), where variants are detected as significant in context with specific traits. These studies identified two of the in-depth characterized 18 coding variants in adrenoceptors as repeatedly statistically significant genetic risk factors - p.Arg389Gly in the β1- and p.Thr164Ile in the β2-adrenoceptor, along with 56 variants in the non-coding regions adjacent to the adrenoceptor gene loci, the functional role of which is largely unknown at present. This chapter summarizes current knowledge on the two coding variants in adrenoceptors that have been consistently validated in GWAS and provides a prospective overview on the numerous non-coding variants more recently attributed to adrenoceptor gene loci.