Measuring the subcellular compartmentalization of viral infections by protein complementation assay

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

The Rice LRR-Like1 Protein YELLOW AND PREMATURE DWARF 1 is Involved in High Light-Induced Leaf Senescence

Senescence is induced by endogenous physiological changes or exogenous stresses in plants. Here, we isolated two alleles of a novel rice (Oryza sativa L.) mutant, yellow and premature dwarf 1 (ypd1). The ypd1 mutants exhibited a yellow and dwarf phenotype from germination, and premature senescence starting at tillering. Moreover, the ypd1 mutants were sensitive to high light, which accelerated cell death and senescence. Consistent with their yellow phenotype, the ypd1 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Trypan blue staining, TUNEL experiments, NBT staining, and DAB staining demonstrated that the ypd1 mutants showed cell death and accumulated reactive oxygen species. Moreover, the ypd1 mutants showed increased expression of senescence-associated genes. Map-based cloning revealed a substitution (G→A) in exon 6 (ypd1-1) and 13 (ypd1-2) of LOC_Os06g13050 that affected splicing and caused premature termination of the encoded protein. YPD1 was preferentially expressed in the leaf and encodes an LRR-Like1 (LRRL1) protein. Complementation, overexpression, and targeted deletion experiments confirmed that the mutations in YPD1 cause the ypd1 phenotype. YPD1 localized on the chloroplast membrane. These findings revealed that the novel rice LRRL1 protein YPD1 affects rice chloroplast development and leaf senescence.

A Bifunctional Cytochrome P450 Enzyme Involved in the O-Dealkylation or N-Dealkoxymethylation of Chloroacetanilide Herbicides in Rhodococcus sp. B2

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant, Due to the large amount of use, its presence in the environment threatens to human health. However, the molecular mechanism of pretilachlor degradation is unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and found to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, 50 mg/L initial substrate, pH 6.98, and 30.1°C. One novel metabolite was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, comprising a gene cluster (EthRABCDB2), was absent in the mutant strain TB2 which could not degrade pretilachlor. The Eth gene system, encodes an AraC/XylS family transcriptional regulator, a ferredoxin reductase, a cytochrome P-450 monooxygenase, a ferredoxin and a 10-kDa unknown protein. Complementation of EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its activity against chloroacetamide herbicides. The codon of EthABCDB2 was optimized, expressed in Escherichia coli, and purified utilizing Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation activity toward alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation activity toward pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation or N-dealkoxymethylation of pretilachlor and propisochlor. And the results provide microbial resource for the remediation of chloroacetamide herbicides-contaminated sites,

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491

Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

In Vivo Methods to Study Protein–Protein Interactions as Key Players in Mycobacterium Tuberculosis Virulence

Studies on protein–protein interactions (PPI) can be helpful for the annotation of unknown protein functions and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher tuberculosis (TB) pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

In Vivo Methods to Study Protein-Protein Interactions as Key Players in Mycobacterium tuberculosis Virulence

Studies on Protein-Protein interactions (PPI) can be helpful for the annotation of unknown protein function and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher TB pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR–GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein–protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR–GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR–GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.