Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Drosophila larval neuromuscular junction's responses to reduction of cAMP in the nervous system

We investigated the effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction, using two fly lines in which cAMP was significantly lower than normal in the nervous system: (a) transgenic flies in which the dunce (dnc) gene product was overexpressed in the nervous system, and (b) flies mutant for the rutabaga gene (rut¹) which have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibited a smaller number of synaptic varicosities. This effect was more pronounced in transgenic larvae, in which the reduction of neural cAMP was more pronounced. Synaptic transmission was also reduced in both cases, as evidenced by smaller excitatory junctional potentials (EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicated almost normal transmitter release properties in transgenic larvae and a modest impairment in rut¹ larvae. Thus, reduction in EJP amplitude in transgenic larvae is primarily due to reduced innervation, while in rut¹ larvae it is attributable to the combined effects of reduced innervation and a mild impairment of transmitter release. We conclude that the major effect of chronically lowered cAMP is reduction of innervation rather than impairment of transmitter release properties. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 1–13, 1999     

Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins'' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein''s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.During the last few decades, the insecticidal toxins produced by Bacillus thuringiensis have been used increasingly in the forms of formulated sprays and transgenic plants for the highly focused biological control of insect pests (29). At the same time, the mechanism by which these proteins form pores in the apical membrane of midgut epithelial cells of targeted insects has been studied extensively (7, 29). In the case of the three-domain Cry toxins, specificity is mostly attributable to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets (16, 27). On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 (17, 20).Several lines of evidence indicate that helices α4 and α5 play a particularly important role in these processes (3). Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were most efficient when α4 and α5 were connected by a segment corresponding to the α4-α5 loop of the toxin (13, 14). A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized (2, 9, 10, 15, 18, 23, 25, 30, 31, 33). Finally, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin (23).Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 (31) but in most of its hydrophilic residues (15). Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or size of the pores but from a reduced pore-forming capacity of the toxin (15, 31). In order to better understand the role of α4 in the mechanism of pore formation, the present study was carried out with a series of previously characterized Cry1Aa mutants in which most of the residues from this helix were replaced by cysteines (15). By subjecting these mutants to circular dichroism (CD), protease sensitivity, pore formation inhibition, and electrophoretic mobility analyses, our data suggest that the mutations in α4 which alter the pore-forming ability of Cry1Aa do so mainly by preventing the proper oligomerization or membrane insertion of the toxin.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.     

Dark diversity in Amazonian stream fish communities: What factors determine species absence along environmental gradients?

1. Species distribution models often fail to predict observed patterns of species diversity, and this is because some species within a regional pool that are tolerant of conditions at a given location may nevertheless be absent from the local community. These missing species have been termed “dark diversity”. In the present study, we investigated which factors explain dark diversity among fish assemblages in Amazonian streams.
2. We sampled 71 streams in areas with different types of land use within two river basins and estimated dark diversity from patterns of species co-occurrence, using Beals’ index, along environmental gradients. From this procedure, taxa are designated as dark diversity components when they are absent from a given stream, but often co-occur with the local species at other streams, indicating similar ecological requirements. We used generalised linear models both to determine whether environmental or landscape variables, connectivity, instream environmental heterogeneity or some combination of these factors explained dark diversity of fishes, and to evaluate whether ecomorphology is associated with the extent to which a species contributes to dark diversity and which specific traits contribute the most to explaining variation in dark diversity.
3. Mean local diversity exceeded observed dark diversity. The magnitude of dark diversity was directly associated with the proportion of secondary forest in the immediate catchment and with the index of proximity to anthropogenic impact. Species that have high affinity for environments with higher current velocity, low swimming ability and that capture food mainly on the surface contributed more to dark diversity, which suggests that swimming ability, habitat preference and aspects related to diet are key predictors of the probability that a given species will be present at locations with suitable habitat.
4. Our findings reinforce the idea that dark diversity results from interactions between species traits and environmental factors, including anthropogenic impacts. Understanding the interplay among environmental factors and species traits that contribute to dark diversity provides targets for improved ecosystem restoration and sustainability of native species assemblages.