Structure‐based analysis and evolution of a monomerized red‐colored chromoprotein from the Olindias formosa jellyfish

GFP‐like chromoproteins (CPs) with non‐fluorescence ability have been used as bioimaging probes. Existing CPs have voids in the optical absorption window which limits their extensibility. The development of new CP color is therefore ongoing. Here, we cloned CPs from the jellyfish, Olindias formosa, and developed a completely non‐fluorescent monomeric red CP, R‐Velour, with an absorption peak at 528 nm. To analyze the photophysical properties from a structural aspect, we determined the crystal structure of R‐Velour at a 2.1 Å resolution. R‐Velour has a trans‐chromophore similar to the green fluorescence protein, Gamillus, derived from the same jellyfish. However, in contrast to the two coplanar chromophoric rings in Gamillus, R‐Velour has a large torsion inducing non‐fluorescence property. Through site‐directed mutagenesis, we surveyed residues surrounding the chromophore and found a key residue, Ser155, which contributes to the generation of four‐color variants with the bathochromic and hypsochromic shift of the absorption peak, ranging from 506 to 554 nm. The recently proposed spectrum shift theory, based on the Marcus–Hush model, supports the spectrum shift of these mutants. These findings may support further development of R‐Velour variants with useful absorption characteristics for bioimaging, including fluorescence lifetime imaging and photoacoustic imaging.     

Improving integrative 3D modeling into low‐ to medium‐resolution electron microscopy structures with evolutionary couplings

Electron microscopy (EM) continues to provide near‐atomic resolution structures for well‐behaved proteins and protein complexes. Unfortunately, structures of some complexes are limited to low‐ to medium‐resolution due to biochemical or conformational heterogeneity. Thus, the application of unbiased systematic methods for fitting individual structures into EM maps is important. A method that employs co‐evolutionary information obtained solely from sequence data could prove invaluable for quick, confident localization of subunits within these structures. Here, we incorporate the co‐evolution of intermolecular amino acids as a new type of distance restraint in the integrative modeling platform in order to build three‐dimensional models of atomic structures into EM maps ranging from 10–14 Å in resolution. We validate this method using four complexes of known structure, where we highlight the conservation of intermolecular couplings despite dynamic conformational changes using the BAM complex. Finally, we use this method to assemble the subunits of the bacterial holo‐translocon into a model that agrees with previous biochemical data. The use of evolutionary couplings in integrative modeling improves systematic, unbiased fitting of atomic models into medium‐ to low‐resolution EM maps, providing additional information to integrative models lacking in spatial data.     

Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

Mammalian cells utilize Akt‐dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low‐density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B‐containing lipoproteins. However, signaling‐controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca²⁺ and calcium/calmodulin‐dependent protein kinase II‐mediated signalings. This response is independent of induction of autophagy. Amino acid stress‐induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet‐induced hypercholesterolemia in heterozygous LDLR‐deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling‐controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.     

Evolutionary conservation of complexins: from choanoflagellates to mice

Complexins are synaptic SNARE complex‐binding proteins that cooperate with synaptotagmins in activating Ca²⁺‐stimulated, synaptotagmin‐dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin‐independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non‐metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca²⁺‐triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.     

A genetic bistable switch utilizing nonlinear protein degradation

Background

Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes.

Results

In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence.

Conclusions

To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.     

An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2

The authors note that P values presented in the original Fig 1A and Appendix Fig S1A were not assessed using a proper statistical analysis method. In contrast to the initially employed two‐group t‐test, a one‐sample one‐tailed t‐test is appropriate here, as the basic null hypothesis is that the proportion of MT FOXL2 mRNA in each AGCT patient is lower than WT {H ₀: WT(%) > MT(%) }. New p values are presented in the corrected Fig 1A and Appendix Fig S1A, which are P < 0.00001 and P < 0.05, respectively. These revised P values did not affect the conclusion drawn.     

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

Background

Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.

Results

Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M⁻¹ cm⁻) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.

Conclusions

The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.     

Genome‐scale metabolic modeling reveals key features of a minimal gene set

Mesoplasma florum, a fast‐growing near‐minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜ 30% of its protein‐coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species‐specific constraints to produce the functional iJL208 genome‐scale model (GEM) of metabolism. Genome‐wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI‐syn3.0 and its parent JCVI‐syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping‐stone toward model‐driven whole‐genome engineering.     

Recent advances using green and red fluorescent protein variants

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.     

Dimensionless parameter predicts bacterial prodrug success

Understanding mechanisms of antibiotic failure is foundational to combating the growing threat of multidrug‐resistant bacteria. Prodrugs—which are converted into a pharmacologically active compound after administration—represent a growing class of therapeutics for treating bacterial infections but are understudied in the context of antibiotic failure. We hypothesize that strategies that rely on pathogen‐specific pathways for prodrug conversion are susceptible to competing rates of prodrug activation and bacterial replication, which could lead to treatment escape and failure. Here, we construct a mathematical model of prodrug kinetics to predict rate‐dependent conditions under which bacteria escape prodrug treatment. From this model, we derive a dimensionless parameter we call the Bacterial Advantage Heuristic (BAH) that predicts the transition between prodrug escape and successful treatment across a range of time scales (1–10⁴ h), bacterial carrying capacities (5 × 10⁴–10⁵ CFU/µl), and Michaelis constants (K_M  = 0.747–7.47 mM). To verify these predictions in vitro, we use two models of bacteria‐prodrug competition: (i) an antimicrobial peptide hairpin that is enzymatically activated by bacterial surface proteases and (ii) a thiomaltose‐conjugated trimethoprim that is internalized by bacterial maltodextrin transporters and hydrolyzed by free thiols. We observe that prodrug failure occurs at BAH values above the same critical threshold predicted by the model. Furthermore, we demonstrate two examples of how failing prodrugs can be rescued by decreasing the BAH below the critical threshold via (i) substrate design and (ii) nutrient control. We envision such dimensionless parameters serving as supportive pharmacokinetic quantities that guide the design and administration of prodrug therapeutics.     

Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own “affinity matrix,” thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active β-galactosidase was obtained in a single step using the above described method.     

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.     

Measuring how two proteins affect each other's net charge in a crowded environment

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein''s charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester‐Staudinger pairs) can be used to mimic crowding by linking two non‐interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine‐acyl “protein charge ladders” and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z _monomer = −0.43 ± 0.01) and α‐lactalbumin (Z _monomer = −4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = −0.04 ± 0.09 upon crowding by this pair (Z _dimer = −5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.     

miR‐AB,a miRNA‐based shRNA viral toolkit for multicolor‐barcoded multiplex RNAi at a single‐cell level

Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss‐of‐function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR‐AB, a new microRNA‐based shRNA (shRNAmir) backbone for simplified, cost‐effective, and error‐proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR‐AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor‐barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single‐cell level.     

Osteoclasts adapt to physioxia perturbation through DNA demethylation

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two‐photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia‐inducible factor activity. We observe that hypoxia decreases ten‐eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen‐dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.     

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.     

Molecular basis of cross‐species ACE2 interactions with SARS‐CoV‐2‐like viruses of pangolin origin

Correction to: The EMBO Journal (2021) 40: e107786. DOI 10.15252/embj.2021107786 | Published online 8 June 2021The authors would like to add three references to the paper: Starr et al and Zahradník et al also reported that the Q498H or Q498R mutation has enhanced binding affinity to ACE2; and Liu et al reported on the binding of bat coronavirus to ACE2.Starr et al and Zahradník et al have now been cited in the Discussion section, and the following sentence has been corrected from:“According to our data, the SARS‐CoV‐2 RBD with Q498H increases the binding strength to hACE2 by 5‐fold, suggesting the Q498H mutant is more ready to interact with human receptor than the wildtype and highlighting the necessity for more strict control of virus and virus‐infected animals”.to“Here, according to our data and two recently published papers, the SARS‐CoV‐2 RBD with Q498H or Q498R increases the binding strength to hACE2 (Starr et al, 2020; Zahradník et al, 2021), suggesting the mutant with Q498H or Q498R is more ready to interact with human receptor than the wild type and highlighting the necessity for more strict control of virus and virus‐infected animals”.The Liu et al citation has been added to the following sentence:“In another paper published by our group recently, RaTG13 RBD was found to bind to hACE2 with much lower binding affinity than SARS‐CoV‐2 though RaTG13 displays the highest whole‐genome sequence identity (96.2%) with the SARS‐CoV‐2 (Liu et al, 2021)”.Additionally, the authors have added the GISAID accession IDs to the sequence names of the SARS‐CoV‐2 in two human samples (Discussion section). To make identification unambiguous, the sequence names have been updated from “SA‐lsf‐27 and SA‐lsf‐37” to “GISAID accession ID: EPI_ISL_672581 and EPI_ISL_672589”.Lastly, the authors declare in the Materials and Methods section that all experiments employed SARS‐CoV‐2 pseudovirus in cultured cells. These experiments were performed in a BSL‐2‐level laboratory and approved by Science and Technology Conditions Platform Office, Institute of Microbiology, Chinese Academy of Sciences.These changes are herewith incorporated into the paper.     

Using near‐infrared reflectance spectroscopy (NIRS) to estimate carbon and nitrogen stable isotope composition in animal tissues

1. Stable isotopes analysis (SIA) of carbon and nitrogen provides valuable information about trophic interactions and animal feeding habits.
2. We used near‐infrared reflectance spectroscopy (NIRS) and support vector machines (SVM) to develop a model for screening isotopic ratios of carbon and nitrogen (δ ¹³C and δ ¹⁵N) in samples from living animals. We applied this method on dried blood samples from birds previously analyzed for δ ¹³C and δ ¹⁵N to test whether NIRS can be applied to accurately estimate isotopic ratios.
3. Our results show a prediction accuracy of NIRS (R ² > 0.65, RMSEP < 0.28) for both δ ¹³C and δ ¹⁵N, representing a 12% of the measurement range in this study.
4. Our study suggests that NIRS can provide a time‐ and cost‐efficient method to evaluate stable isotope ratios of carbon and nitrogen when substantial differences in δ ¹³C or δ ¹⁵N are expected, such as when discriminating among different trophic levels in diet.