Cytosolic Protein Research Articles

Functional transfer of integrin co-receptor CD98hc by small extracellular vesicles improves wound healing in vivo.

Extracellular vesicles (EVs) mediate intercellular communication. EVs are composed of a lipid bilayer and contain cytosolic proteins and RNAs. Studies highlight EVs striking functions in cell-cell crosstalk. Here, we found that small EVs can transfer functional signaling molecules through their lipid bilayer and participate in skin homeostasis. We identified a transmembrane protein CD98hc (a.k.a. SLC3A2), an integrin co-receptor (Itgb1 and Itgb3), implicated in epidermis homeostasis via its capacity in regulating extracellular matrix, as an important mediator of EV-based intercellular communication in vivo. We first demonstrated that healthy dermal fibroblasts produced and secreted EVs bearing characteristic of exosome-like small EVs (sEVs). We show that CD98hc, Itgb1 co-receptor, is present at the surface of sEVs, transferred and stabilized at the plasma membrane. The transferred complex is functional on recipient cells both in vitro and in vivo. Indeed, treatment with sEVs from WT, but not KO cells rescued migratory defects observed either in CD98hc KO dermal fibroblasts or in keratinocytes in vitro. Furthermore, injection of sEVs at the margins of wound in impaired wound healing mouse models (epidermal CD98hc KO mice exhibiting healing defect and elderly mice) improved wound closure in vivo. CD98hc complex transferred from sEVs remained stabilized at least 7 days after injection. Thus, our findings reveal that in vivo treatment with sEVs containing integrin co-receptor CD98hc could improve multiple skin afflictions.

Expression Profile of Thymidine Kinase Genes in Cervical Squamous Cell Carcinoma Confirmed by Various Detection Methods.

Thymidine kinases (TKs) are key enzymes involved in DNA synthesis and repair, with alterations in their expression associated with various cancers. Thymidine kinase 1 (TK1) and TK2 are cytosolic enzyme proteins that catalyze the addition of a gamma-phosphate group to thymidine. The existing literature on TK1 in cervical squamous cell carcinoma (CESC) fails to address the clinical role of TK1 overexpression and its possible molecular mechanism in CESC. The clinical significance of TK2 in CESC is also unknown. The objective was to explore the differential expression, clinical significance, and molecular mechanisms of TK1 and TK2 in CESC. The researchers collected global high-throughput data, extracted the expression levels of TK1 and TK2, and calculated the integrated standardized mean difference (SMD) and summarized receiver's operating characteristics (sROC) of TK1 or TK2 mRNA to investigate the expression profiles of TK genes fully and objectively in 918 CESC tissues and 360 control tissues. In-house tissue microarrays for immunohistochemical testing were used to verify the protein level of TK1 in 62 CESC tissues and control tissues. The growth effect of TK1 and TK2 in CESC cell lines was assessed using Chronos dependency scores derived from CRISPR knockout screen in the Achilles project. We also analyzed the potential mechanism of TK genes by studying the relationship between TK gene expression and immune infiltration, gene alternations as well as the related signal pathways. The various detection methods employed all confirmed that the TK1 expression is upregulated and TK2 is downregulated in CESC tissues (SMD: 2.44, 95% confidence interval (CI): 1.36 - 3.51, area under curve (AUC): 0.88, 95% CI: 0.85 - 0.90; SMD: -0.69, 95% CI: -1.25 to -0.14, AUC: 0.75, 95% CI: 0.71 - 0.78). Inhibition of TK1 expression by CRISPR knockout had negative influence on the biological functions of 11 CESC cell lines. The expression of TK2 was negatively correlated with the malignant progression of CESC. Expression of TK genes showed significant association with the immune infiltration of macrophages, CD4+ T cells, and neutrophils. Genes related with TK1 or TK2 were involved in pathways related to DNA replication, proteasome, and homologous recombination. Clinically, these findings suggest that the differential expression of TK1 and TK2 could serve as potential biomarkers, as well as therapeutic targets for personalized treatment strategies in CESC patients.

NADPH oxidase 4-SH3 domain-containing YSC84-like 1 complex participates liver inflammation and fibrosis.

There is growing evidence that NADPH oxidase 4 (Nox4) in hepatocytes contributes to liver inflammation and fibrosis during the development of metabolic dysfunction-associated steatohepatitis (MASH). However, how Nox4 is regulated and leads to liver pathogenesis is unclear. Our previous studies showed that the cytosolic protein SH3 domain-containing Ysc84-like 1 (SH3YL1) regulates Nox4 activity. Here, we asked whether SH3YL1 also participates in liver inflammation and fibrosis during MASH development. We generated that whole body SH3YL1 knockout (SH3YL1-/-), Nox4 knockout (Nox4-/-) mice, and the hepatocyte-specific SH3YL1 conditional knockout (Alb-Cre/SH3YL1fl/fl) mice were fed a methionine/choline-deficient (MCD) diet to induce liver inflammation and fibrosis in pathogenesis of MASH. Palmitate-stimulated primary SH3YL1- and Nox4-deficient hepatocytes and hepatic stellate cells (HSCs) did not generate H2O2. While the liver of MCD diet-fed wild type (WT) mice demonstrated elevated 3-nitrotyrosine as a protein oxidation and 4-hydroxynonenal adducts as a lipid oxidation and increased liver inflammation, hepatocyte apoptosis, and liver fibrosis, these events were markedly reduced in SH3YL1-/-, Nox4-/-, and Alb-Cre/SH3YL1fl/fl mice. The MCD diet-fed WT mice also showed elevated hepatocyte expression of SH3YL1 protein. Similarly, liver biopsies from MASH patients demonstrated strong hepatocyte SH3YL1 protein expression, whereas hepatocytes from patients with steatosis weakly expressed SH3YL1 and histologically normal patient hepatocytes exhibited very little SH3YL1 expression. The Nox4-SH3YL1 complex in murine hepatocytes elevates their H2O2 production, which promotes the liver inflammation, hepatocyte apoptosis, and liver fibrosis that characterize MASH. This axis may also participate in MASH in humans.

An avoidance segment resolves a lethal nuclear-mitochondrial targeting conflict during ribosome assembly.

The correct sorting of nascent ribosomal proteins from the cytoplasm to the nucleus or to mitochondria for ribosome production poses a logistical challenge for cellular targeting pathways. Here we report the discovery of a conserved mitochondrial avoidance segment (MAS) within the cytosolic ribosomal protein uS5 that resolves an evolutionary lethal conflict between the nuclear and mitochondrial targeting machinery. MAS removal mistargets uS5 to the mitochondrial matrix and disrupts the assembly of the cytosolic ribosome. The resulting lethality can be rescued by impairing mitochondrial import. We show that MAS triages nuclear targeting by disabling a cryptic mitochondrial targeting activity within uS5 and thereby prevents fatal capture by mitochondria. Our findings identify MAS as an essential acquisition by the primordial eukaryote that reinforced organelle targeting fidelity while developing an endosymbiotic relationship with its mitochondrial progenitor.

Differential protein and mRNA cargo loading into engineered large and small extracellular vesicles reveals differences in in vitro and in vivo assays.

Extracellular vesicles (EV) represent an advanced platform for genetic material and protein delivery, particularly when they are loaded through the so-called endogenous loading method. This study investigates the differences between large EV (lEV) and small EV (sEV) obtained from genetically engineered C2C12 myoblasts overexpressing two different model biomolecules. Erythropoietin (EPO) is a secretory protein with anti-inflammatory, angiogenic and hematopoietic effects, while TGL is a chimeric cytosolic protein containing green fluorescent protein (GFP) and luciferase, used for imaging. We compared these EV subtypes in terms of protein and nucleic acid loading, intercellular cargo transfer capacity, and subsequent functional effects both in vitro and in vivo. Our results demonstrated that lEV exhibited higher protein and mRNA cargo content than sEV, which also translated into increased intercellular cargo transfer capacity, even when dosing according to the constitutive sEV and lEV secretion ratio (10,1). Indeed, we showed that, although receptor cells successfully internalized both EV subtypes, cells treated with lEV displayed stronger intracellular luciferase signals and higher EPO protein secretion compared to those treated with sEV. In terms of functional effects, both EV subtypes exerted anti-inflammatory and antioxidant effects in lipopolysaccharide-activated macrophages, as well as angiogenic effects in human umbilical vein endothelial cells. Finally, in vivo studies evidenced that subcutaneously administered lEV led to a more significant increase in hematocrit levels and red blood cell counts than sEV. Taken together, these findings suggest that the protein and mRNA cargo differ between endogenously loaded EV subtypes, and that this variation in cargo loading leads to differences in their functional outcomes. Therefore, the choice of EV subtype could be critical for optimizing EV-based delivery strategies for biologic drugs.

Monoaminergic neurotransmitters are bimodal effectors of tau aggregation.

Neurotransmitters (NTs) mediate trans-synaptic signaling, and disturbances in their levels are linked to aging and brain disorders. Here, we ascribe an additional function for NTs in mediating intracellular protein aggregation by interaction with cytosolic protein fibrils. Cell-based seeding experiments revealed monoaminergic NTs as inhibitors of tau. Seeding is a disease-relevant mechanism involving catalysis by fibrils, leading to the aggregation of proteins in Alzheimer's disease and other neurodegenerative diseases. Chemotyping small molecules with varied backbone structures revealed determinants of aggregation inhibitors and catalysts. Among those identified were monoaminergic NTs. Dose titrations revealed bimodal effects indicative of fibril disaggregation, with aggregation catalysis occurring at low ratios of NTs and inhibited seeding ensuing at higher concentrations. Bimodal effects by NTs extend from in vitro systems to dopaminergic neurons, suggesting that pharmacotherapies that modify intracellular NT levels could shape the neuronal protein aggregation environment.

A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588.

Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyric acid. Butyrate is a four-carbon, short-chain fatty acid (SCFA) which has multiple health benefits. Many microbial products of pharmaceutical or industrial interest, such as butyrate, are produced in low quantities due to rate-limiting steps in their metabolic pathway, including low abundance or low activity of one or more enzymes in the pathway. By identifying the former, appropriate enzymes can be over-expressed to increase product yields, however, methods to determine these enzymes are laborious. To improve butyrate production in C. butyricum probiotic strain, CBM588, a novel rapid genome-proteome approach was deployed. First, whole genome sequencing was performed and the 8 genes involved in butyrate production identified on the chromosome. Second, the relative abundance of these enzymes was investigated by liquid chromatography-mass spectrometry (LC-MS) analysis of total cytosolic proteins from early stationary phase cultures. Phosphotransbutyrylase (Ptb), butyrate kinase (Buk) and crotonase (Crt) were found to be the least abundant. Over-expression episomally of the corresponding genes individually or of the ptb-buk bicistron led to significant increases in butyrate titre per density of culture from 10 to 24 hours, compared to the wild type. Our findings pave the way for over-expressing these genes chromosomally to generate a recombinant probiotic with improved butyrate production and potentially enhanced gut health properties.

O-GlcNAcylated FTO promotes m6A modification of SOX4 to enhance MDS/AML cell proliferation

Fat mass and obesity-associated protein (FTO) was the first m6A demethylase identified, which is responsible for eliminating m6A modifications in target RNAs. While it is well-established that numerous cytosolic and nuclear proteins undergo O-GlcNAcylation, the possibility of FTO being O-GlcNAcylated and its functional implications remain unclear. This study found that a negative correlation between FTO expression and O-GlcNAcylation in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The decreased O-GlcNAcylation on FTO can result in diminished m6A modification of SRY-related high mobility group box 4 (SOX4). This led to the promotion of cell apoptosis and inhibition of cell proliferation in MDS/AML. The O-GlcNAcylation of FTO stabilized SOX4 transcripts in an m6A-dependent manner, resulting in increased AKT and MAPK phosphorylation and decreased cell apoptosis. Inhibiting FTO O-GlcNAcylation significantly slowed AML progression in vitro, a finding supported by clinical data in MDS/AML patients. In conclusion, our study highlights the crucial role of FTO O-GlcNAcylation in RNA m6A methylation and the progression of MDS/AML, thereby providing a potential therapeutic avenue for these formidable diseases.

Guanidyl-rich highly branched poly(β-amino ester)s for the delivery of dual CRISPR ribonucleoprotein for efficient large DNA fragment deletion.

Gene editing technologies, particularly clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, have revolutionized the ability to modify gene sequences in living cells for therapeutic purposes. Delivery of CRISPR/Cas ribonucleoprotein (RNP) is preferred over its DNA and RNA formats in terms of gene editing effectiveness and low risk of off-target events. However, the intracellular delivery of RNP poses significant challenges and necessitates the development of non-viral vectors. Our previous study has demonstrated that phenyl guanidine (PG) group modified linear poly(β-amino ester)s (PAEs) can facilitate CRISPR/Cas9 RNP mediated gene knockout in HeLa cells. Here, we further investigated the utilization of highly branched PAEs (HPAEs) with PG groups (HPAE-PG) for efficient delivery of cytosolic protein and CRISPR/Cas9 RNP complexes, while also examining the influence of branching units and branching ratios on the delivery process. The efficiency of HPAE-PG/RNP transfection for large DNA fragment deletion was assessed using a dual sgRNA-guided approach to delete exon 80 of the human COL7A1 gene, which harbors mutations associated with dystrophic epidermolysis bullosa (DEB). Our findings demonstrate that HPAE-PG/RNP successfully induced a deletion of 56 base pairs (exon 80) within COL7A1 in both HEK cells and keratinocytes derived from recessive DEB patients. This study highlights the potential of HPAE-PG as a non-viral vector for large DNA fragment deletion, emphasizing the importance of branching factors of HPAEs in optimizing CRISPR RNP delivery for therapeutic applications in genetic disorders.

Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation.

A low-molecular-weight compound whose structure strikes a fine balance between hydrophobicity and hydrophilicity may form coacervates via liquid-liquid phase separation in an aqueous solution. These coacervates may encapsulate and convoy proteins across the plasma membrane into the cell. However, releasing the cargo from the vehicle to the cytosol is challenging. Here, we address this issue by designing phase-separating coacervates, which are disassembled by the bioorthogonal Staudinger reaction. We constructed and selected triphenylphosphine-based compounds that formed phase-separated coacervates in an aqueous solution. Reacting the coacervates with azides resulted in microdroplet dissolution, so they received the name Staudinger Reaction-Responsive Coacervates, SR-Coa. SR-Coa could encapsulate proteins, including antibodies, and translocate them across the plasma membrane into the cell. Further treatment of the cell with ethyl azidoacetate induced the cargo dispersion from the puncta to the cytosolic distribution. We showcased an application of the SR-Coa/ethyl azidoacetate system in facilitating the translocation of the EGFR/antibody complex into the cell, which induced EGFR degradation via the TRIM21-dependent pathway both in vitro and in vivo. Besides the membrane protein EGFR, this system could also degrade endogenous protein EZH2. Taken together, here we report a strategy of controlling molecular coacervates by a bioorthogonal reaction in the cell for cytosolic protein delivery and demonstrate its use in promoting targeted protein degradation via the proteasome-dependent pathway.

Pathophysiological Role of Neutrophil Extracellular Traps in Diet-Induced Obesity and Metabolic Syndrome in Animal Models.

The global pandemic of obesity poses a serious health, social, and economic burden. Patients living with obesity are at an increased risk of developing noncommunicable diseases or to die prematurely. Obesity is a state of chronic low-grade inflammation. Neutrophils are first to be recruited to sites of inflammation, where they contribute to host defense via phagocytosis, degranulation, and extrusion of neutrophil extracellular traps (NETs). NETs are web-like DNA structures of nuclear or mitochondrial DNA associated with cytosolic antimicrobial proteins. The primary function of NETosis is preventing the dissemination of pathogens. However, neutrophils may occasionally misidentify host molecules as danger-associated molecular patterns, triggering NET formation. This can lead to further recruitment of neutrophils, resulting in propagation and a vicious cycle of persistent systemic inflammation. This scenario may occur when neutrophils infiltrate expanded obese adipose tissue. Thus, NETosis is implicated in the pathophysiology of autoimmune and metabolic disorders, including obesity. This review explores the role of NETosis in obesity and two obesity-associated conditions-hypertension and liver steatosis. With the rising prevalence of obesity driving research into its pathophysiology, particularly through diet-induced obesity models in rodents, we discuss insights gained from both human and animal studies. Additionally, we highlight the potential offered by rodent models and the opportunities presented by genetically modified mouse strains for advancing our understanding of obesity-related inflammation.

Hsf1 is essential for proteotoxic stress response in smyd1b-deficient embryos and fish survival under heat shock.

Molecular chaperones play critical roles in post-translational maintenance in protein homeostasis. Previous studies have shown that loss of Smyd1b function results in defective myofibril organization and dramatic upregulation of heat shock protein gene (hsp) expression in muscle cells of zebrafish embryos. To investigate the molecular mechanisms and functional importance of this stress response, we characterized changes of gene expression in smyd1b knockdown and knockout embryos using RNA-seq. The results showed that the top upregulated genes encode mostly cytosolic heat shock proteins. Co-IP assay revealed that the upregulated cytosolic Hsp70s associate with myosin chaperone UNC45b which is critical for myosin protein folding and sarcomere assembly. Strikingly, several hsp70 genes also display muscle-specific upregulation in response to heat shock-induced stress in zebrafish embryos. To investigate the regulation of hsp gene upregulation and its functional significance in muscle cells, we generated heat shock factor 1 (hsf-/-) knockout zebrafish mutants and analyzed hsp gene expression and muscle phenotype in the smyd1b-/-single and hsf1-/-;smyd1b-/- double-mutant embryos. The results showed that knockout of hsf1 blocked the hsp gene upregulation and worsened the muscle defects in smyd1b-/- mutant embryos. Moreover, we demonstrated that Hsf1 is essential for fish survival under heat shock (HS) conditions. Together, these studies uncover a correlation between Smyd1b deficiency and the Hsf1-activated heat shock response (HSR) in regulating muscle protein homeostasis and myofibril assembly and demonstrate that the Hsf1-mediated hsp gene upregulation is vital for the survival of zebrafish larvae under thermal stress conditions.

Nonenzymatic lysine d-lactylation induced by glyoxalase II substrate SLG dampens inflammatory immune responses

Immunometabolism is critical in the regulation of immunity and inflammation; however, the mechanism of preventing aberrant activation-induced immunopathology remains largely unclear. Here, we report that glyoxalase II (GLO2) in the glycolysis branching pathway is specifically downregulated by NF-κB signaling during innate immune activation via tristetraprolin (TTP)-mediated mRNA decay. As a result, its substrate S-D-lactoylglutathione (SLG) accumulates in the cytosol and directly induces d-lactyllysine modification of proteins. This nonenzymatic lactylation by SLG is greatly facilitated by a nearby cysteine residue, as it initially reacts with SLG to form a reversible S-lactylated thiol intermediate, followed by SN-transfer of the lactyl moiety to a proximal lysine. Lactylome profiling identifies 2255 lactylation sites mostly in cytosolic proteins of activated macrophages, and global protein structure analysis suggests that proximity to a cysteine residue determines the susceptibility of lysine to SLG-mediated d-lactylation. Furthermore, lactylation is preferentially enriched in proteins involved in immune activation and inflammatory pathways, and d-lactylation at lysine 310 (K310) of RelA attenuates inflammatory signaling and NF-κB transcriptional activity to restore immune homeostasis. Accordingly, TTP-binding site mutation or overexpression of GLO2 in vivo blocks this feedback lactylation in innate immune cells and promotes inflammation, whereas genetic deficiency or pharmacological inhibition of GLO2 restricts immune activation and attenuates inflammatory immunopathology both in vitro and in vivo. Importantly, dysregulation of the GLO2/SLG/d-lactylation regulatory axis is closely associated with human inflammatory phenotypes. Overall, our findings uncover an immunometabolic feedback loop of SLG-induced nonenzymatic d-lactylation and implicate GLO2 as a promising target for combating clinical inflammatory disorders.

Glial cells improve Parkinson's disease by modulating neuronal function and regulating neuronal ferroptosis.

The main characteristics of Parkinson's disease (PD) are the loss of dopaminergic (DA) neurons and abnormal aggregation of cytosolic proteins. However, the exact pathogenesis of PD remains unclear, with ferroptosis emerging as one of the key factors driven by iron accumulation and lipid peroxidation. Glial cells, including microglia, astrocytes, and oligodendrocytes, serve as supportive cells in the central nervous system (CNS), but their abnormal activation can lead to DA neuron death and ferroptosis. This paper explores the interactions between glial cells and DA neurons, reviews the changes in glial cells during the pathological process of PD, and reports on how glial cells regulate ferroptosis in PD through iron homeostasis and lipid peroxidation. This opens up a new pathway for basic research and therapeutic strategies in Parkinson's disease.

Discovery of PRMT5 N-Terminal TIM Barrel Ligands from Machine-Learning-Based Virtual Screening.

Protein arginine methyltransferase 5 (PRMT5), which symmetrically dimethylates cytosolic and nuclear proteins, has been demonstrated as an important cancer therapeutic target. In recent years, many advanced achievements in PRMT5 inhibitor development have been made. Most PRMT5 inhibitors in the clinical trial focus on targeting the C-terminal catalytic domain, whereas developing small molecules to interrupt the PRMT5/pICLn (methylosome subunit) protein-protein interface is also of great importance for inhibiting PRMT5. Here, we describe a machine-learning-based virtual screening method and use this novel pipeline to screen small-molecule inhibitors of the PRMT5/pICLn interaction. 18 compounds were manually selected for experimental testing. One compound, Z319334062, showed surface plasmon resonance-binding affinity to the target (K D = 21.5 μM) and dose-dependently inhibited symmetric dimethylation levels in patient-derived glioblastoma cell lines.

Intracellular protein-lipid interactions drive presynaptic assembly prior to neurexin recruitment.

Neurexin cell-adhesion molecules regulate synapse development and function by recruiting synaptic components. Here, we uncover a mechanism for presynaptic assembly that precedes neurexin recruitment, mediated by interactions between cytosolic proteins and membrane phospholipids. Developmental imaging in C.elegans reveals that the intracellular active zone protein SYD-1 accumulates at nascent presynapses prior to its binding partner neurexin. Combining molecular dynamics simulations to model intrinsic interactions between SYD-1 and lipid bilayers with biochemical and invivo validation of these predictions, we find that PIP2-interacting residues in the SYD-1 C2 domain are required for active zone assembly. Genetic perturbation of a PIP2-generating enzyme disrupts synaptic SYD-1 accumulation, while the PIP2-interacting domain of mammalian RIM1 can compensate for the SYD-1 C2 domain, suggesting functional homology between these proteins. Finally, we propose that the evolutionarily conserved γ-neurexin isoform represents a minimal neurexin sequence that stabilizes nascent presynaptic assemblies, potentially a core function of this isoform.

Using synthetic biology to express nitrogenase biosynthesis pathway in rice and to overcome barriers of nitrogenase instability in plant cytosol.

Engineering nitrogen fixation in cereals could reduce usage of chemical nitrogen fertilizers. Here, a nitrogenase biosynthesis pathway comprising 13 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV nifS nifU groES groEL) was introduced into rice by transforming multigene vectors and subsequently by sexual crossing between transgenic rice plants. Genome sequencing analysis revealed that 13 nif genes in F4 hybrid rice lines L12-13 and L8-17 were inserted at two loci on rice chromosome 1. Eleven nitrogen fixation (Nif) proteins were produced and stable NifDK tetramer was formed in rice cytosol. NifH in rice cytosol was unstable and NifH-S18 was found to be a key residue that conferred susceptibility to proteinase degradation. NifH variants with Fe protein activity and resistance to plant endoproteinase cleavage were obtained. This study provides an efficient approach for introducing multiple nif genes into plants and also helps to pre-evaluate the stability of prokaryotic proteins in plant cytosol.

Advanced Method for the In Vivo Measurements of Lysophospholipid Translocation Across the Inner (Cytoplasmic) Membrane of Escherichia coli.

Phospholipid translocation occurs ubiquitously in biological membranes and primarily is protein catalyzed. Lipid flippases mediate the net translocation of specific phospholipids from one leaflet of a membrane to the other. In the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria, lysophospholipid translocase (LplT) and cytosolic bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase (Aas) form a glycerophospholipid regeneration system, which is capable of facilitating rapid retrograde translocation of lyso forms of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) but not exogenous (host-derived) phosphatidylcholine (PC) across the IM of Gram-negative diderm (two-membraned) bacteria in consequential order lyso-PE=lyso-PG >>lysophosphatidic acid (lyso-PA) >>lyso-PC. Although several flippases that bind and move non-glycerophosphatidyl lipids across the IM are characterized in Gram-negative bacteria, LplT appears to be the first example of a bacterial protein capable of facilitating the rapid translocation of monoacylated glycerophospholipids. On the cytoplasmic surface, Aas restores the lysophospholipids to their diacyl forms with comparable efficiency but excludes any exogenous monoacylated lipid species. This coupled remodeling enzyme tandem provides an effective means to examine substrate specificity of lipid regeneration and lysophospholipid transport per se across the membrane. The current chapter describes two distinct but complementary methods for the measurement of lysophospholipid transport across membranes using Escherichia coli spheroplasts.

Pinus radiata cDNA Library for the Screening of Phytophthora Effector Protein Targets in Yeast.

Yeast two-hybrid library screening enables the discovery of novel protein-protein interactions. Identifying cytosolic host proteins targeted by host-translocated Phytophthora effector proteins relies on the mRNA amount, quality, and composition used to prepare the yeast two-hybrid cDNA library. Here we describe the steps required for the preparation of a Pinus radiata cDNA library optimized for Phytophthora effector target screening in yeast. This method may also be applied to other host plants and pathogen species.

Afadin Sorts Different Retinal Neuron Types into Accurate Cellular Layers.

Neurons use cell-adhesion molecules (CAMs) to interact with other neurons and the extracellular environment: the combination of CAMs specifies migration patterns, neuronal morphologies, and synaptic connections across diverse neuron types. Yet little is known regarding the intracellular signaling cascade mediating the CAM recognitions at the cell surface across different neuron types. In this study, we investigated the neural developmental role of Afadin1-4, a cytosolic adapter protein that connects multiple CAM families to intracellular F-actin. We introduced the conditional Afadin mutant5 to an embryonic retinal Cre, Six3-Cre6-8. We reported that the mutants lead to the scrambled retinal neuron distribution, including Bipolar Cells (BCs), Amacrine Cells (ACs), and retinal ganglion cells (RGCs), across three cellular layers of the retina. This scrambled pattern was first reported here at neuron-type resolution. Importantly, the mutants do not display deficits for BCs, ACs, or RGCs in terms of neural fate specifications or survival. Additionally, the displayed RGC types still maintain synaptic partners with putative AC types, indicating that other molecular determinants instruct synaptic choices independent of Afadin. Lastly, there is a significant decline in visual function and mis-targeting of RGC axons to incorrect zones of the superior colliculus, one of the major retinorecipient areas. Collectively, our study uncovers a unique cellular role of Afadin in sorting retinal neuron types into proper cellular layers as the structural basis for orderly visual processing.