Transmembrane Sequence Research Articles

Genome-wide computational analysis of the dirigent gene family in Solanum lycopersicum

BackgroundDirigent (DIR) genes play a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. They are required to produce lignans, which are a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential role in plant development. DIR genes are implicated in the process of lignification and protect plants from environmental stresses, including biotic and abiotic stresses. Nevertheless, no research has been performed on the DIR gene family in Solanum lycopersicum. This study provides detailed information on the DIR gene family in S. lycopersicum.Methods and resultsThe conserved domain analysis, phylogenetic analysis, evolutionary adaptation, cis-acting elements, proteomic analysis, signal peptide detection, transmembrane potential analysis, sequence identity and similarity analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of 31 potential DIR genes were studied. All these analyses provide a deep understanding of DIR genes in the S. lycopersicum genome that will provide a useful reference for further functional analysis of the DIR genes in S. lycopersicum.ConclusionThis research provides an in-depth and comprehensive explanation of the detailed process and structural characterization of DIR genes in the genome of S. lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This work will provide valuable information for identifying DIR genes in higher plants and support future research on the DIR gene family.

A vaccine candidate based on baculovirus displaying chikungunya virus E1-E2 envelope confers protection against challenge in mice.

Chikungunya fever is a re-emerging mosquito-borne disease caused by the chikungunya virus (CHIKV) and produces acute arthritis that can progress to chronic disease with arthralgia. The first approved live-attenuated chikungunya vaccine has only recently become available for use in humans in the USA, but the access in endemic regions remains unmet. Here, we exploited the baculovirus display technology to develop a vectored vaccine candidate that exposes the CHIKV membrane proteins E1 and E2 on the baculovirus surface. Using recombinant baculovirus as vector vaccines has both productive and regulatory advantages: they are safe for handling and easy to produce in high titers and are non-pathogenic and non-replicative in mammals but have strong adjuvant properties by inducing humoral and cellular immune responses. CHIKV E1 and E2 envelope proteins with their own signal and transmembrane sequences were expressed on the surface of budded baculovirus virions. Immunization of C57BL/6 mice with non-adjuvanted recombinant baculovirus induced IgG antibodies against E2 with a predominant IgG2c subtype, neutralizing antibodies and a specific IFN-γ CD8+ T-cell response. Immunization with a second dose significantly boosted the antibody response, and mice immunized with two doses of the vaccine candidate were completely protected against challenge with CHIKV showing no detectable viremia or signs of disease. Altogether, baculovirus display of CHIKV envelope proteins served as an efficient vaccine platform against CHIKV.IMPORTANCEThe global spread of chikungunya virus (CHIKV) has disproportionately impacted the Americas that experienced a fourfold increase in 2023 in cases and deaths compared with the same period in 2022. The disease is characterized by acute fever and debilitating joint pain that can become chronic. Despite the socioeconomic burden related to the high morbidity rates of CHIKV infection, a vaccine for CHIKV is currently approved only in the USA. Vaccines are the most effective preventive measure against viral diseases, and advances in the development of different vaccine platforms such as nucleic acids and viral vectors have prompted the rapid deployment of vaccines to contain the COVID-19 pandemic. Here, we report the use of baculovirus display as a strategy for the design of a novel vaccine that provides sterilizing immunity in a mouse model of chikungunya disease. Our results encourage further research regarding the potential of baculovirus as platforms for human vaccine design.

Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amendable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed “nanodysferlins”, designed to eliminate several C2 domains, specifically C2 domains D, E and F; B, D and E; and B, D, E and F. We also generated a variant by replacing 8 amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, 3 of them supported membrane repair after laser wounding while all 4 bound the membrane repair protein, TRIM72/MG53, similar to wild type dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

Structural bioinformatics studies of glutamate transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Glutamate transporters play key roles in nervous physiology by modulating excitatory neurotransmitter levels, when malfunctioning, involving in a wide range of neurological and physiological disorders. However, integral transmembrane proteins including the glutamate transporters remain notoriously difficult to study, due to their localization within the cell membrane. Here we present the structural bioinformatics studies of glutamate transporters and their water-soluble variants generated through QTY-code, a protein design strategy based on systematic amino acid substitutions. These include 2 structures determined by X-ray crystallography, cryo-EM, and 6 predicted by AlphaFold2, and their predicted water-soluble QTY variants. In the native structures of glutamate transporters, transmembrane helices contain hydrophobic amino acids such as leucine (L), isoleucine (I), and phenylalanine (F). To design water-soluble variants, these hydrophobic amino acids are systematically replaced by hydrophilic amino acids, namely glutamine (Q), threonine (T) and tyrosine (Y). The QTY variants exhibited water-solubility, with four having identical isoelectric focusing points (pI) and the other four having very similar pI. We present the superposed structures of the native glutamate transporters and their water-soluble QTY variants. The superposed structures displayed remarkable similarity with RMSD 0.528Å-2.456Å, despite significant protein transmembrane sequence differences (41.1%->53.8%). Additionally, we examined the differences of hydrophobicity patches between the native glutamate transporters and their QTY variants. Upon closer inspection, we discovered multiple natural variations of L->Q, I->T, F->Y and Q->L, T->I, Y->F in these transporters. Some of these natural variations were benign and the remaining were reported in specific neurological disorders. We further investigated the characteristics of hydrophobic to hydrophilic substitutions in glutamate transporters, utilizing variant analysis and evolutionary profiling. Our structural bioinformatics studies not only provided insight into the differences between the hydrophobic helices and hydrophilic helices in the glutamate transporters, but they are also expected to stimulate further study of other water-soluble transmembrane proteins.

In Silico Analyses of Vertebrate G-Protein-Coupled Receptor Fusions United With or Without an Additional Transmembrane Sequence Indicate Classification into Three Groups of Linkers.

Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)-(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI-L-SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates.

Identification of a novel protein localized to the crystalloid of the Plasmodium ookinete

Reducing Plasmodium parasite transmission via the mosquito vector is a promising strategy for malaria control and elimination in endemic regions. In the mosquito midgut after the ingestion of an infected blood meal, malaria parasite gametes egress from erythrocytes and fertilize to develop into motile ookinetes that traverse midgut epithelial cells and transform into oocysts adjacent the basal lamina. Plasmodium ookinetes and young oocysts possess a unique organelle called the crystalloid; which has a honeycomb-like matrix structure and is indicated to be involved in sporozoite formation and maturation. In this study, we identified a novel crystalloid protein, PY17X_1113800, that is exclusively expressed in developing ookinetes. The protein possesses a signal peptide sequence, but lacks a transmembrane domain or GPI anchor signal sequence, as well as predicted adhesive domains which are characterisitic of many crystalloid proteins. The protein is highly conserved across the phylum Apicomplexa and within the greater clade Alveolata, such as Vitrella and the ciliates Paramecium and Tetrahymena, but is absent in cryptosporidia.

Abstract 1319: In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors

Abstract Introduction: Current generation of adoptive cell therapy relies on ex vivo engineering of immune cells with tumor-targeting chimeric antigen receptors. In addition to manufacturing challenges, ex vivo engineered cells such as CAR-NK and CAR-T show limited tumor infiltration and persistency within tumor microenvironment. A method leveraging mRNA-based delivery for in-vivo engineering of these cells could address these issues but has not been established. Conventional mRNA/LNP when infused systemically result in significant accumulation in liver tissue and the CAR has no immune cell specificity in their expression or function. Method: Many immune receptors are heterodimeric complexes composed of a ligand binding subunit (e.g. CD16 and TCR) and a signaling adaptor containing Immunoreceptor Tyrosine-based Activation Motif (ITAM) (e.g. FcRγ and CD3ζ). Notably, the cell surface expression of the ligand binding subunit often depends on the expression of the ITAM adaptor. Based on this we fused HER2-targeting scFv to multiple activating receptors whose expression is restricted to NK cells and T cells, and evaluated their expression and anti-tumor function. Results: We developed a series of Natural Cytotoxic Receptor (NCR)-based in vivo CAR constructs by fusing HER2-targeting scFv to the native extracellular and transmembrane sequences of the receptor NKp30, NKp44 and NKp46, These CARs engage signaling adaptor FcRγ or DAP12 expressed in NK cells, and exhibit potent tumoricidal activity against HER2+ ovarian cancer cells as well as mediate tumor-induced cytokine and chemokine response. Using Huh7 liver cell as a model for off-target expression in hepatocytes, we found that NKp44 and NKp46-based CAR requires their cognate signaling adaptors for stable cell surface expression. We also showed that similar strategy can be used to design receptors for selective expression on the surface of T cells utilizing CD3ε chain fused with tumor targeting scFv. Conclusion: These results demonstrated the feasibility of engineering primary NK cells with CARs based on the NCR family of receptors NKp44 and NKp46, whose expression requires the immune-restricted signaling adaptors. These CAR-NK receptors do not contain intracellular signaling motifs but can engage and activate their signaling natural adaptors to initiate tumor killing and cytokine response. An mRNA/LNP based delivery of NKp44-based CAR for cancer treatment is currently under clinical development. Citation Format: Neha Diwanji, Daniel Getts, Yuxiao Wang. In vivo programming of natural killer cells and T cells using mRNA delivered cytotoxic chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1319.

Structural bioinformatics studies of serotonin, dopamine and norepinephrine transporters and their AlphaFold2 predicted water-soluble QTY variants and uncovering the natural mutations of L->Q, I->T, F->Y and Q->L, T->I and Y->F.

Monoamine transporters including transporters for serotonin, dopamine, and norepinephrine play key roles in monoaminergic synaptic signaling, involving in the molecular etiology of a wide range of neurological and physiological disorders. Despite being crucial drug targets, the study of transmembrane proteins remains challenging due to their localization within the cell membrane. To address this, we present the structural bioinformatics studies of 7 monoamine transporters and their water-soluble variants designed using the QTY code, by systematically replacing the hydrophobic amino acids leucine (L), valine (V), isoleucine (I) and phenylalanine (F) with hydrophilic amino acids (glutamine (Q), threonine (T) and tyrosine (Y). The resulting QTY variants, despite significant protein transmembrane sequence differences (44.27%-51.85%), showed similar isoelectric points (pI) and molecular weights. While their hydrophobic surfaces significantly reduced, this change resulted in a minimal structural alteration. Quantitatively, Alphafold2 predicted QTY variant structures displayed remarkable similarity with RMSD 0.492Å-1.619Å. Accompanied by the structural similarities of substituted amino acids in the context of 1.5Å electron density maps, our study revealed multiple QTY and reverse QTY variations in genomic databases. We further analyzed their phenotypical and topological characteristics. By extending evolutionary game theory to the molecular foundations of biology, we provided insights into the evolutionary dynamics of chemically distinct alpha-helices, their usage in different chemotherapeutic applications, and open possibilities of diagnostic medicine. Our study rationalizes that QTY variants of monoamine transporters may not only become distinct tools for medical, structural, and evolutionary research, but these transporters may also emerge as contemporary therapeutic targets, providing a new approach to treatment for several conditions.

Mechanistic Elucidation of the Tumor-Promoting Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 in B-Cell Receptor Signaling in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a highly aggressive and incurable type of B-cell non-Hodgkin's lymphoma. B cell receptor (BCR) signaling plays an important role in the pathogenesis of MCL; however, the underlying mechanisms are unknown. Our genome-wide screens identified that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1/CC1) is a central component of BCR signaling and is essential for MCL survival in vitro and in vivo. Among the twelve alternatively spliced human CC1 isoforms, we found that CC1-4L, the longest CC1 isoform is overexpressed in MCL. Structurally, it has the N-terminal domain and three type 2 constant ectodomains, a transmembrane sequence and a signaling cytoplasmic domain with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs or Y493/520). These ITIMs of CC1 can inhibit signals in T cells, NK cells, neutrophils and granulocytes. However, its role in B cells and BCR signaling is not clear. In this study, we have elucidated a novel mechanistic function of CC1 in promoting oncogenic BCR signaling in MCL. First, we studied the role of CC1 in lipid raft formation, which is one of the early events during BCR signaling activation. To study this, we used three panels of MCL lines, Jeko-1-NTC (non-template control), Jeko-1-gCC1 (CC1-knockout) and Jeko-1-gCC1-4L (CC1-knockout restored with CC1-4L isoform). Using high-resolution confocal microscopy, we observed that CC1-expressing Jeko-1 MCL cell line showed significantly increased lipid raft formation when compared to CC1-knockout cells. However, CC1-deficient cells restored with the CC1-4L isoform exhibited increased lipid raft assembly. Further, we also observed that CC1-expressing MCL lines showed increased cytoskeletal assembly, which was observed via increased F-actin and filamin A staining in CC1-expressing MCL lines when compared to CC1-knockout cells. Second, we analyzed the phosphorylation levels of proteins involved in the BCR signaling cascade in a panel of genetically engineered MCL lines, i.e., Jeko-1-NTC, Jeko-1-gCC1, Jeko-1-gCC1-4L, Jeko-1-gCC1-4S (CC1-knockout restored with CC1-4S or short cytoplasmic tail isoform) and Jeko-1-gCC1-mutants (CC1-knockout cells restored with N-terminal domain deletion mutant and ITIM-mutant or YY/FF 493/520). We observed that Jeko-1-NTC and Jeko-1-gCC1-4L cell lines showed significantly increased phosphorylation levels of BCR signaling components such as phospho-SRC (Y416), phospho-SYK (Y319), phospho-AKT (S473) and phospho-BTK (Y223) when compared to Jeko-1-gCC1, Jeko-1-gCC1-4S and Jeko-1-gCC1 mutant MCL lines. This shows that CC1 positively regulates or activates oncogenic BCR signaling in MCL and the N-terminal domain and cytoplasmic tail are required for this function. Next, we analyzed whether CC1 could interact with the BCR signaling components such as CD79B, LYN and SYK using immunoprecipitation (IP) and proximity ligation assay (PLA) in MCL lines. Further, we analyzed the kinetics of BCR activation in order to infer the role of SHP-1 binding to the ITIMs of CC1. Our IP and PLA experiments demonstrated that in Jeko-1 and Mino MCL lines, at the peak (i.e., at 5 and 15 min) of BCR activation, there was significantly increased interaction between CC1 and the BCR-pathway components (CD79B, LYN and SYK), and this interaction subsided by 30 min of IgM activation. However, in the case of CC1 and SHP-1, the interaction was stable throughout the time points and increased by 30 min of IgM activation. These interactions were disrupted in the CC1-N-terminal domain deletion and ITIM mutant MCL lines when compared to the CC-4L expressing MCL line. Finally, we analyzed the interaction between CD79B and SYK in CC1-4L, CC1-ITIM mutant and N-domain deletion mutant expressing MCL lines. Using PLA, we observed increased interaction between CD79B and SYK in CC1-4L expressing cell line compared to other cell lines indicating that functional CC1 is required for the recruitment of SYK to CD79B. In summary, CC1 stabilizes lipid rafts following BCR engagement by anchoring to F-actin and Filamin A. CC1 recruits and activates LYN and SYK at the BCR complex at the peak of BCR activation and subsequently recruits SHP-1 for signal downregulation. These activities of CC1 require intact ITIMs and the N-terminal ligand-binding domain. Hence, CC1 drives oncogenic BCR signaling in MCL and forms an important candidate for MCL immunotherapy.

Cloning and functional characterization of porcine AACS revealing the regulative roles for fat deposition in pigs.

Fat deposition is a quantitative trait controlled by multiple genes in pigs. Using transcriptome sequencing, we previously reported that AACS is differentially expressed in the subcutaneous fat tissue of Dingyuan pigs with divergent backfat thickness. Therefore, with the aim of further characterizing this gene and its protein, we cloned the entire 3286-bp mRNA sequence of the porcine AACS, and the encoded AACS protein is a hydrophilic protein without a signal peptide or transmembrane sequence. Our findings suggested that among various tissues and pig breeds, AACS was highly expressed in subcutaneous fat. We have identified three completely linked SNP loci in the AACS gene: A-1759C, C-1683T, and A-1664G. The double luciferase activity test in the 5' flanking region indicated that the flanking region of AACS contained several active regulatory elements. The three linked SNPs that were identified in one of the critical active elements, and might serve as important molecular markers regulating backfat thickness. Finally, we observed that AACS overexpression inhibited the proliferation and differentiation of subcutaneous preadipocytes. Collectively, our results suggest that AACS inhibits subcutaneous fat deposition in pigs. This study provides a new molecular marker for understanding the mechanism of porcine fat deposition.

Molecular Structure, Biochemical Functions, Genetics, and Emerging Clinical Relevance of Glucose Transporters

In the human body, glucose acts as a major energy-producing fuel and regulator of energy homeostasis, enzyme functions, and gene transcription. The selective permeability of the lipid bilayer structure of the cell membrane makes it mandatory for glucose to require transport proteins for its transit into the cells. These include solute carrier integral membrane proteins such as glucose transporters (GLUTs) and sodium-glucose transporters. GLUTs belong to the major facilitator superfamily with a 12 transmembrane spanner topology, with GLUT1–13 sharing the same transmembrane sequence but variable transmembrane loops and terminal cytoplasmic ends of carbon and nitrogen. Phylogenetic analysis classifies GLUTs into three classes, with each class showing an affinity for a specific substrate. The tightly coupled relationship between glucose homeostasis and the nearly ubiquitous GLUTs has led to the investigation of their diverse roles in embryonic development, adult physiology, and clinical disorders including but not limited to inborn errors, diabetes mellitus, metabolic syndrome, and cancers. The current review is pivoted around the studies focusing on the structure and functions of members of the GLUT family, their chromosomal and organ-specific distribution, as well as the current evidence of their clinical implications and prospective therapeutic roles, specifically in cancers and metabolic disorders. The literature for the present work was retrieved from databases including Google Scholar, Web of Science, and PubMed.

Plant cadmium resistance 10 enhances tolerance to toxic heavy metals in poplar

Toxic heavy metals originating from human activities have caused irreversible harm to the environment. Toxic heavy metal ions absorbed by crop plants can seriously threaten human health. Therefore, decreasing heavy metal contents in crop plants is an urgent need. The plant cadmium resistance protein (PCR) is a heavy metal ion transporter. In this study, PePCR10 was cloned from Populus euphratica. Bioinformatics analyses revealed its transmembrane structure and gene sequence motifs. The transcript profile of PePCR10 was analyzed by RT-qPCR, and its transcript levels increased under toxic heavy metal (cadmium, lead, aluminum) treatments. Subcellular localization analyses in tobacco cells revealed that PePCR10 localizes at the plasma membrane. Compared with wild type (WT), PePCR10-overexpressing lines showed significantly higher values for plant height, root length, fresh weight, and dry weight under heavy metal stress. Electrolyte leakage, nitroblue tetrazolium staining, and chlorophyll fluorescence analyses indicated that Cd/Al tolerance in PePCR10-overexpressing lines was stronger than that in WT. The Cd/Al contents were lower in the PePCR10-overexpressing lines than in WT under Cd/Al stress. Our results show that PePCR10 can reduce the heavy metal content in poplar and enhance its Cd/Al tolerance. Hence, PePCR10 is a candidate genetic resource for effectively reducing heavy metal accumulation in crops.

Arabidopsis transmembrane proteins regulate protein turnover as signaling molecules during internalization by means of ubiquitination

<p>This study attempts to characterize the structure, function, and other features of Arabidopsis thaliana based on its transmembrane protein amino acid sequence and assess its potential as a drug target and ubiquitination site. The transmembrane protein belongs to the Arabidopsis plant, but little is known about the structure-function of the protein, among others. We predicted the structure and transmembrane domains of the transmembrane protein by different bioinformatic tools, and the prediction results showed that its three poorly conserved transmembrane domains could be used as potential drug targeting sites. Among them, lysine ubiquitination sites play an important role in the recycling of plant membrane proteins, and we found the existence of ubiquitination sites by predicting the ubiquitination sites of transmembrane proteins, and this could be a new research direction in the future.<strong></strong></p>

Implication of the Transmembrane Domain in the Interleukin 10 Receptor Platform Oligomerisation.

Interleukin 10 (IL-10) exerts anti-inflammatory and immune regulatory roles through its fixation to the IL-10 receptor (IL-10R). The two subunits (IL-10Rα and IL-10Rβ) organise themselves to form a hetero-tetramer to induce the activation of the transcription factor STAT3. We analysed the activation patterns of the IL-10R, especially the contribution of the transmembrane (TM) domain of the IL-10Rα and IL-10Rβ subunits, as evidence accumulates that this short domain has tremendous implications in receptor oligomerisation and activation. We also addressed whether targeting the TM domain of IL-10R with peptides mimicking the TM sequences of the subunits translates into biological consequences. The results illustrate the involvement of the TM domains from both subunits in receptor activation and feature a distinctive amino acid crucial for the interaction. The TM peptide targeting approach also appears to be suitable for modulating the activation of the receptor through its action on the dimerization capabilities of the TM domains and thereby constitutes a potential new strategy for the modulation of the inflammation in pathologic contexts.

Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.

Identification of a novel heat shock protein 33 of Pythium insidiosum from the first Chinese skin and subcutaneous Pythiosis

ObjectiveTo predict and analyze the structure and function of heat shock protein 33 by bioinformatics analysis. MethodsThe physical and chemical properties, hydrophilicity (hydrophobicity), transmembrane structure, secondary and tertiary structure, subcellular localization, signal peptide and glycosylation, phosphorylation sites, B cell, helper T (Th) cell epitopes of Hsp protein isolated from Pythium insidiosum were analyzed by corresponding bioinformatics software. ResultsHsp33 was composed of 608 amino acids, and the molecular formula was C2182H3538N624O654S16. Hsp33 was a hydrophilic unstable protein. The protein had no transmembrane domain and signal peptide sequence. Subcellular localization analysis shows that the amino acid was a nuclear protein;α-helix, β-sheet, β-turn and random coil accounted for 43.59%, 14.47%, 5.26% and 36.68%, respectively. There were 14 glycosylation sites and 48 phosphorylation sites. It has T cell and B cell dominant epitopes. TLR9 had the best affinity to HSP33 based on its binding affinity. ConclusionThis study lays the foundation for future research on vaccine construct and also provides clues to explore the pathogenic mechanism of HSP33-TLR9 signal pathway for Pythium insidiosum.

Activity of EGFR transmembrane region variants indicates specific transmembrane dimers are not required for EGFR activity.

The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase that mediates cell proliferation and differentiation events during development and maintenance of complex organisms. Formation of specific, ligand-dependent EGFR dimers is a key step in stimulating EGFR signaling, and crystal structures of active, dimeric forms of isolated EGFR extracellular regions and kinase domains have revealed much about how dimer interactions regulate EGFR activity. The nature and role of the transmembrane region in regulating EGFR activity remains less clear, however. Proposed roles for the transmembrane region range from nonspecific but energetically favorable interactions to specific transmembrane dimer conformations being associated with active, inactive, or activity-modulated states of EGFR. To investigate the role of specific transmembrane dimers in modulating EGFR activity we generated thirteen EGFR variants with altered transmembrane sequences designed to favor or disfavor specific types of transmembrane region interactions. We show using FRET microscopy that EGFR transmembrane regions have an intrinsic propensity to associate in mammalian cell membranes that is counteracted by the extracellular region. We show using cell-based assays that each of the EGFR transmembrane variants except the Neu variant, which results in constitutive receptor phosphorylation, is able to autophosphorylate and stimulate phosphorylation of downstream effectors Erk and Akt. Our results indicate that many transmembrane sequences, including polyleucine, are compatible with EGFR activity and provide no evidence for specific transmembrane dimers regulating EGFR function.

T cell and B cell antigen receptors share a conserved core transmembrane structure

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR's homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.