GFP Fusion Protein Research Articles

Epimedium flavonoids mitigate proteotoxicity and extend healthspan via DAF-16 inC. elegans

Objective: Epimedium flavonoids (EF), the raw extract of medicinal herb Epimedium, have been shown to have broad beneficial effects in the elderly including those with neurodegeneration. The goal of this study is to investigate whether EF is protective against proteotoxicity and whether it extends healthspan in C. elegans.Methods: Animals were treated with EF supplemented in the growth medium. Two C. elegans models of human proteotoxic disease, CL4176 expressing an aggregated amyloid-[Formula: see text] (1–42) peptide (A[Formula: see text]1–42) and AM140 expressing a polyglutamine (polyQ) protein, were exploited to test the anti-proteotoxicity of EF. Proteotoxicity-induced paralysis in CL4176 and AM140 was evaluated. Lifespan, stress resistance, and locomotion were tested in wild-type N2 C. elegans. Lifespan assays were also performed in CF1038, a daf-16 null mutant strain. DAF-16 nuclear translocation was analyzed in TJ356, a strain expressing a functional DAF-16::GFP fusion protein. The mRNA levels of downstream targets of DAF-16 were measured by qPCR.Results: EF significantly reduced A[Formula: see text]1–42- and polyQ-induced paralysis in CL4176 and AM140, indicating the anti-proteotoxic potency of EF. EF significantly extended the lifespan and promoted stress resistance and locomotion in N2, demonstrating a healthspan extension effect of EF. DAF-16 nuclear localization and its downstream targets, sod-3 and hsp-16.2 mRNA levels, were significantly elevated with EF. EF did not increase the lifespan of daf-16 null mutant CF1038, revealing a DAF-16-dependent mechanism of EF effect on lifespan.Conclusion: We found that EF, a natural extract from a widely used medicinal herb Epimedium, protects against proteotoxicity and extends the healthspan via DAF-16 in C. elegans. Our work may provide molecular insights into the clinical benefits of EF and Epimedium.

Designing an ELP-intein system: toward a more realistic outlook

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.

ZGrad is a nanobody-based degron system that inactivates proteins in zebrafish.

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

Surface acoustic waves enable rotational manipulation of Caenorhabditis elegans.

Controllable, precise, and stable rotational manipulation of model organisms is valuable in many biomedical, bioengineering, and biophysics applications. We present an acoustofluidic chip capable of rotating Caenorhabditis elegans (C. elegans) in both static and continuous flow in a controllable manner. Rotational manipulation was achieved by exposing C. elegans to a surface acoustic wave (SAW) field that generated a vortex distribution inside a microchannel. By selectively activating interdigital transducers, we achieved bidirectional rotation of C. elegans, namely counterclockwise and clockwise, with on-demand switching of rotation direction in a single chip. In addition to continuous rotation, we also rotated C. elegans in a step-wise fashion with a step angle as small as 4° by pulsing the signal duration of SAW from a continuous signal to a pulsed signal down to 1.5 ms. Using this device, we have clearly imaged the dopaminergic neurons of C. elegans with pdat-1:GFP expression, as well as the vulval muscles and muscle fibers of the worm with myo-3::GFP fusion protein expression in different orientations and three dimensions. These achievements are difficult to realize through conventional (i.e., non-confocal) microscopy. The SAW manipulations did not detectably affect the health of the model organisms. With its precision, controllability, and simplicity in fabrication and operation, our acoustofluidic devices will be well-suited for model organism studies.

Identification of the evolutionarily conserved nuclear envelope proteins Lem2 and MicLem2 in Tetrahymena thermophila.

Lem2 family proteins, i.e. the LAP2-Emerin-MAN1 (LEM) domain-containing nuclear envelope proteins, are well-conserved from yeasts to humans, both of which belong to the Opisthokonta supergroup. However, whether their homologs are present in other eukaryotic phylogenies remains unclear. In this study, we identified two Lem2 homolog proteins, which we named as Lem2 and MicLem2, in a ciliate Tetrahymena thermophila belonging to the SAR supergroup. Lem2 was localized to the nuclear envelope of the macronucleus (MAC) and micronucleus (MIC), while MicLem2 was exclusively localized to the nuclear envelope of the MIC. Immunoelectron microscopy revealed that Lem2 in T. thermophila was localized to both the inner and outer nuclear envelopes of the MAC and MIC, while MicLem2 was mostly localized to the nuclear pores of the MIC. Molecular domain analysis using GFP-fused protein showed that the N-terminal and luminal domains, including the transmembrane segments, are responsible for nuclear envelope localization. During sexual reproduction, enrichment of Lem2 occurred in the nuclear envelopes of the MAC and MIC to be degraded, while MicLem2 was enriched in the nuclear envelope of the MIC that escaped degradation. These findings suggest the unique characteristics of Tetrahymena Lem2 proteins. Our findings provide insight into the evolutionary divergence of nuclear envelope proteins.

Aged CAR T Cells Exhibit Enhanced Cytotoxicity and Effector Function but Shorter Persistence and Less Memory-like Phenotypes

▪[Introduction]CD19 chimeric antigen receptor (CAR) T cell therapies have been approved by the FDA for children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adults with r/r large B-cell lymphoma. Recent reports about long-term follow-up of CD19 CAR T cell therapy in B-ALL (Maude et. al. NEJM 2018, Park et. al. NEJM 2018) suggest that the median event-free survival of children and young adult patients is longer than that of adult patients (Over 11 months versus 6.1 months). The reason for the difference between survival of pediatric and adult patient is unclear, but we hypothesize it is due to age-related changes in the T cells collected from patients. Therefore, we compared the function of CAR T cells derived from young or aged mice.[Methods]Young C57BL/6J (B6) mice (6-12 weeks) and aged B6 mice (³ 72 weeks) were used as donors for CAR T cell preparation. Four types of mouse specific CD19 CAR encoded GFP fusion proteins were evaluated with all having the same anti-CD19 scFv and CD8 hinge and transmembrane domains but differing in their intracellular domain (m19Δz: lacks the CD3ξ signaling domain, m19z: CD3ξ signaling domain only, m1928z: CD28 and CD3ξ signaling domains, m19-humBBz: 4-1BB and CD3ξ signaling domains).[Results]T cells isolated from the spleen of aged B6 mice were significantly fewer than those of young B6 mice. However, CAR transduction efficiency, viability and yield were similar between young and aged CAR T cells for each CAR group. All groups of aged CAR T cells predominate with CD8+ and effector-like phenotypes at the expense of CD4+ and memory-like phenotypes after CD19+ artificial antigen presenting cell (aAPC) stimulation (Fig. 1A-1B). Furthermore, compared to CAR T cells derived from young mice, aged CAR T cells (m19z, m1928z and m19BBz) exhibited superior cytotoxicity in a real-time cell analysis for CD19+ aAPC killing (Fig. 1C). Using our immune competent in vivo murine model, aged CAR T cells were short-lived and expanded poorly despite their superior in vitro cytotoxicity. To evaluate for potential mechanisms involving preferential production of effector-like CAR T cells from aged mice we performed gene-expression, as well as single cell secretory polyfunctional analyses. While the polyfunctional strength index (PSI) of CD8+ aged CAR T cells was higher for aged CAR T cells, the increased score was due mostly to abundant secretion of a chemokine (Fig. 1D). Furthermore, the RNA-DESeq analysis demonstrated increased expression of chemokines and perturbation of the EOMES/TBET transcription factor axis. RNA-DESeq also suggested that young CAR T cells were highly active in cell proliferation and cell differentiation whereas aged CAR T cells upregulated gene expression pathways that regulated responses to stimulus and exocytosis.[Conclusions]CAR T cells derived from aged mice exhibited enhanced cytotoxicity but shorter persistence and less memory-like phenotypes. Our results suggest that the difference of clinical outcome between younger patients and older patients may be due to an age-dependent CAR T cell phenotype that is reflected by its unique gene expression pattern, secretory profile, and/or transcription factor balance. In our future directions we are extending these observations to human CAR T cells and identifying potential methods to improve the function of aged CAR T cells. [Display omitted] DisclosuresDavila:Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

P110α and p110β isoforms of PI3K are involved in protection against H2O2 induced oxidative stress in cancer cells.

Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110β, two ubiquitously expressed isoforms of PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110β and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110β isoforms in protecting MCF-7 breast cancer cells against oxidative stress. We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis. To know whether p110α and p110β are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110β proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels. We report the involvement of p110α and p110β isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.

The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an animal model of Parkinson\u2019s disease

Accumulation of alpha-synuclein (ASYN) in neurons and other CNS cell types may contribute to the underlying pathology of synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and Multiple Systems Atrophy (MSA). In support of this hypothesis for PD, ASYN immunopositive aggregates are a prominent pathological feature of PD, and mutations and gene multiplications of human wild type (WT) ASYN cause rare familial autosomal-dominant forms of PD. Targeted therapeutics that reduce the accumulation of ASYN could prevent or slow the neurodegenerative processes in PD and other synucleinopathies. NPT200-11 is a novel small molecule inhibitor of ASYN misfolding and aggregation. The effects of NPT200-11 on ASYN neuropathology were evaluated in animal models over expressing human alpha synuclein. Longitudinal studies using retinal imaging in mice expressing a hASYN::GFP fusion protein revealed that 2 months of once daily administration of NPT200-11 (5 mg/kg IP) resulted in a time-dependent and progressive reduction in retinal ASYN pathology. The effects of NPT200-11 on ASYN pathology in cerebral cortex and on other disease-relevant endpoints was evaluated in the Line 61 transgenic mouse model overexpressing human wild type ASYN. Results from these studies demonstrated that NPT200-11 reduced alpha-synuclein pathology in cortex, reduced associated neuroinflammation (astrogliosis), normalized striatal levels of the dopamine transporter (DAT) and improved motor function. To gain insight into the relationship between dose, exposure, and therapeutic benefit pharmacokinetic studies were also conducted in mice. These studies demonstrated that NPT200-11 is orally bioavailable and brain penetrating and established target plasma and brain exposures for future studies of potential therapeutic benefit.

Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein

Glutathione peroxidase‐like thioredoxin peroxidase (PfTPxGl) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum. Apicoplast trafficking signals usually consist of N‐terminal signal and transit peptides, but the trafficking signal of PfTPxGl appears to exhibit important differences. As transfection is a protracted process in P. falciparum, we expressed the N terminus of PfTPxGl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii, in order to dissect its trafficking signals. We show that PfTPxGl possesses an N‐terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma—this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered PfTPxGl targeting in Toxoplasma, in Plasmodium. This study has demonstrated that PfTPxGl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.

MiR-129-5p inhibits autophagy and apoptosis of H9c2 cells induced by hydrogen peroxide via the PI3K/AKT/mTOR signaling pathway by targeting ATG14

Ischemic heart disease (IHD) is a significant cause of cardiovascular diseases. MicroRNAs (miRNAs) have been thought to be critical regulators in the heart diseases. The present study was aimed to investigate the effect of miR-129-5p on the autophagy and apoptosis by targeting ATG14 as well as how miR-129-5p worked through the PI3K/AKT/mTOR signaling pathway in H2O2-induced H9c2 cells. H9c2 cells were induced by H2O2, after which the expression of miR-129-5p was decreased. Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-129-5p in H9c2 cells. In addition, the expression of miR-129-5p and ATG14 were overexpressed or down-regulated after transfection. The transfection efficiency was verified by qRT-PCR. Cell viability, cell apoptosis, and the expression of autophagy and apoptosis-related proteins were determined by CCK-8, flow cytometry and western blotting, respectively. Furthermore, GFP fusion protein analysis was used to detect the expression level of LC3II which was related to autophagy. As a result, cell viability was decreased and cell autophagy was increased in H2O2-induced H9c2 cells. MiR-129-5p overexpression inhibited cell injury caused by H2O2 in H9c2 cells which was certified by the increased cell viability and decreased cell autophagy and apoptosis. In addition, ATG14 was demonstrated to be a target of miR-129-5p which inhibited cell injury by down-regulation of ATG14. Moreover, phosphorylation of PI3K/AKT/mTOR pathway was activated by miR-129-5p overexpression or ATG14 inhibition to alleviate the autophagy and apoptosis in H2O2-induced H9c2 cells. In conclusion, this study indicated that miR-129-5p inhibited autophagy and apoptosis in H2O2-induced H9c2 cells partly by down-regulation of ATG14 through the activation of PI3K/AKT/mTOR pathway.

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.

Identification and Localization of Peroxisomal Biogenesis Proteins Indicates the Presence of Peroxisomes in the Cryptophyte Guillardia theta and Other "Chromalveolates".

Peroxisomes are single-membrane-bound organelles with a huge metabolic versatility, including the degradation of fatty acids (β-oxidation) and the detoxification of reactive oxygen species as most conserved functions. Although peroxisomes seem to be present in the majority of investigated eukaryotes, where they are responsible for many eclectic and important spatially separated metabolic reactions, knowledge about their existence in the plethora of protists (eukaryotic microorganisms) is scarce. Here, we investigated genomic data of organisms containing complex plastids with red algal ancestry (so-called “chromalveolates”) for the presence of genes encoding peroxins—factors specific for the biogenesis, maintenance, and division of peroxisomes in eukaryotic cells. Our focus was on the cryptophyte Guillardia theta, a marine microalga, which possesses two phylogenetically different nuclei of host and endosymbiont origin, respectively, thus being of enormous evolutionary significance. Besides the identification of a complete set of peroxins in G. theta, we heterologously localized selected factors as GFP fusion proteins via confocal and electron microscopy in the model diatom Phaeodactylum tricornutum. Furthermore, we show that peroxins, and thus most likely peroxisomes, are present in haptophytes as well as eustigmatophytes, brown algae, and alveolates including dinoflagellates, chromerids, and noncoccidian apicomplexans. Our results indicate that diatoms are not the only “chromalveolate” group devoid of the PTS2 receptor Pex7, and thus a PTS2-dependent peroxisomal import pathway, which seems to be absent in haptophytes (Emiliania huxleyi) as well. Moreover, important aspects of peroxisomal biosynthesis and protein import in “chromalveolates”are highlighted.

The regulatory subunit phr2AB of Dictyostelium discoideum phosphatase PP2A interacts with the centrosomal protein CEP161, a CDK5RAP2 ortholog.

phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.

Sphingosine Kinase Activates the Mitochondrial Unfolded Protein Response and Is Targeted to Mitochondria by Stress

SUMMARYThe mitochondrial unfolded protein response (UPRmt) is critical for maintaining mitochondrial protein homeostasis in response to mitochondrial stress, but early steps in UPRmt activation are not well understood. Here, we report a function for SPHK-1 sphingosine kinase in activating the UPRmt in C. elegans. Genetic deficiency of sphk-1 in the intestine inhibits UPRmt activation, whereas selective SPHK-1 intestinal overexpression is sufficient to activate the UPRmt. Acute mitochondrial stress leads to rapid, reversible localization of SPHK-1::GFP fusion proteins with mitochondrial membranes before UPRmt activation. SPHK-1 variants lacking kinase activity or mitochondrial targeting fail to rescue the stress-induced UPRmt activation defects of sphk-1 mutants. Activation of the UPRmt by the nervous system requires sphk-1 and elicits SPHK-1 mitochondrial association in the intestine. We propose that stress-regulated mitochondrial recruitment of SPHK-1 and subsequent S1P production are critical early events for both cell autonomous and cell non-autonomous UPRmt activation.

Isolation and Expression Analysis of PeDWF1 in Phyllostachys edulis

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carriere) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.

Systemic Upregulation of MTP2- and HMA2-Mediated Zn Partitioning to the Shoot Supplements Local Zn Deficiency Responses.

Low bioavailable concentrations of the micronutrient zinc (Zn) limit agricultural production on 40% of cultivated land. Here, we demonstrate that plant acclimation to Zn deficiency involves systemic regulation. Physiological Zn deficiency of Arabidopsis thaliana shoots results in increased root transcript levels of the membrane transport protein-encoding genes METAL TRANSPORT PROTEIN2 (MTP2) and HEAVY METAL ATPASE2 (HMA2), which are unresponsive to the local Zn status of roots. MTP2 and HMA2 act additively in the partitioning of Zn from roots to shoots. Chimeric GFP fusion proteins of MTP2 complement an mtp2 mutant and localize in the endoplasmic reticulum (ER) membrane of the outer cell layers from elongation to root hair zone of lateral roots. MTP2 restores Zn tolerance in a hypersensitive yeast mutant. These results are consistent with cell-to-cell movement of Zn toward the root vasculature inside the ER-luminal continuum through the desmotubules of plasmodesmata, under Zn deficiency. The previously described Zn deficiency response comprises transcriptional activation of target genes, including ZINC-REGULATED TRANSPORTER IRON-REGULATED TRANSPORTER PROTEIN genes ZIP4 and ZIP9, by the F-group bZIP transcription factors bZIP19 and bZIP23. We show that ZIP4 and ZIP9 respond to the local Zn status in both roots and shoots, in contrast to the systemic regulation identified here. Our findings are relevant for crop management and improvement toward combating human nutritional Zn deficiency that affects 30 to 50% of the world's population.

A GFP-fusion coupling FACS platform for advancing the metabolic engineering of filamentous fungi

BackgroundThe filamentous fungus Trichoderma reesei, the most widely used cellulase producer, also has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high value-added products. However, such applications are thwarted by the time-consuming metabolic engineering processes (design–build–test–learn cycle) for T. reesei, resulted from (i) the spore separation-mediated purification as the multinucleate hyphae, (ii) transformant screening for high expression levels since unavailable of episomal expression system, and (iii) cases of inexpressible heterologous proteins.ResultsIn this study, a GFP-fusion coupled fluorescence-activated cell sorting (FACS) platform was established to speed up the build and test process of the DBTL cycle, by enabling rapid selection for expressible heterologous genes and bypassing both laborious spore separation and transformant screening. Here, the feasibility of flow cytometry in analyzing and sorting T. reesei cells harboring GFP-fused expressible protein was proven, as well as the application of the platform for constitutive promoter strength evaluation. As a proof-of-concept, the platform was employed to construct the first T. reesei strain producing fatty alcohol, resulting in up to 2 mg hexadecanol being produced per gram biomass. Pathway construction was enabled through rapid selection of functional fatty acyl-CoA reductase encoding gene Tafar1 from three candidate genes and strains with high expression level from spore pools. As a result of using this method, the total costed time for the build and test cycle using T. reesei, subsequently, reduced by approx. 75% from 2 months to 2 weeks.ConclusionThis study established the GFP-fusion coupling FACS platform and the first filamentous fungal fatty alcohol-producing cell factory, and demonstrated versatile applications of the platform in the metabolic engineering of filamentous fungi, which can be harnessed to potentially advance the application of filamentous fungi in lignocellulose-based biorefinery.

Atomic insights into the genesis of cellular filaments by globular proteins.

Self-assembly of proteins into filaments, such as actin and tubulin filaments, underlies essential cellular processes in all three domains of life. The early emergence of filaments in evolutionary history suggests that filament genesis might be a robust process. Here we describe the fortuitous construction of GFP fusion proteins that self-assemble as fluorescent polar filaments in Escherichia coli. Filament formation is achieved by appending as few as 12 residues. Crystal structures reveal that the protomers each donate an appendage to fill a groove between two following protomers along the filament. This exchange of appendages resembles runaway domain swapping but is distinguished by higher efficiency because monomers cannot competitively bind their own appendages. Ample evidence of this “runaway domain coupling” mechanism in nature suggests it could facilitate the evolutionary pathway from globular protein to polar filament, requiring a minimal extension of protein sequence and no significant refolding.

Late ribosomal protein localization in Arabidopsis thaliana differs to that in Saccharomyces cerevisiae.

Ribosome biogenesis is essential for cellular function and involves rRNA synthesis, rRNA processing and modification, and ribosomal protein assembly. Ribosome biogenesis factors and small nucleolar RNA assist these events. Ribosomal maturation takes place in the nucleolus, the nucleoplasm, and the cytosol in a coordinated and controlled manner. For example, some ribosomal proteins are thought to be assembled in the cytoplasm based on the observations in Saccharomyces cerevisiae. Here, we used cellular fractionation to demonstrate that cleavage of the 20S intermediate, the precursor to mature 18S rRNA, does not occur in the nucleoplasm of Arabidopsis thaliana. It most likely occurs in the cytoplasm. Further, we verified the proposed localization of RPS10e, RPS26e, and RPL24a/b in the nucleus and RPP1 in the nucleolus of A. thaliana by ribosome profiling, immunofluorescence, and analysis of the localization of GFP fusion proteins. Our results suggest that the order of events during ribosomal protein assembly in the ribosome biogenesis pathway differs between plants and yeast.

Glycerol-3-phosphate dehydrogenase (GPDH) gene family in Zea mays L.: Identification, subcellular localization, and transcriptional responses to abiotic stresses.

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the formation of glycerol-3-phosphate, and plays an essential role in glycerolipid metabolism and in response to various stresses in different species. In this study, six ZmGPDH genes were obtained by a thorough search against maize genome, and designated as ZmGPDH1-6, respectively. The structural and evolutionary analyses showed that the ZmGPDHs family had typical conserved domains and similar protein structures as the known GPDHs from other plant species. ZmGPDHs were divided into NAD+-dependent type A form (ZmGPDH1-5) and FAD-dependent type B form (ZmGPDH6) based on their N-terminal sequences. Four full length ZmGPDHs were fused with GFP fusion proteins, and their subcellular localization was determined. ZmGPDH1 and ZmGPDH3 were located to the cytosol and mainly recruited to the surface of endoplasmic reticulum (ER), whereas ZmGPDH4 and ZmGPDH5 were located in the chloroplast. The transcriptional analysis of the ZmGPDHs in different maize tissues revealed relatively high level of transcripts accumulation of ZmGPDHs in roots and early stage developing seeds. Furthermore, we examined the transcriptional responses of the six GPDH genes in maize under various abiotic stresses, including salt, drought, alkali and cold, and significant induction of ZmGPDHs under osmotic stresses was observed. Together, this work will provide useful information for deciphering the roles of GPDHs in plant development and abiotic stress responses.