SARS‐CoV‐2–host proteome interactions for antiviral drug discovery

Treatment options for COVID‐19, caused by SARS‐CoV‐2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS‐CoV‐2–host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS‐CoV‐2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP‐MS) and the complementary proximity‐based labeling MS method (BioID‐MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS‐CoV‐2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image‐based drug screen with infectious SARS‐CoV‐2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein–protein interactions.     

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment

Amino Acids - The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal–epidermal junction and connects the cytoplasmic...     

Dispersion of vendace eggs and larvae around potential nursery areas reveals their reproductive strategy

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LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.     

Within species variation in host plant quality and movement behavior of Lygus rugulipennis nymphs

We studied movement behavior of the polyphagous herbivore Lygus rugulipennis Poppius (Heteroptera: Miridae) on wheat (Triticum aestivum) that had been cultivated in pots using three different levels of nitrogen fertilization. The probability of moving for fourth instar nymphs, and the time spent moving by mobile fourth and fifth instar nymphs increased with the nitrogen fertilization level of the pot. The nitrogen fertilization level of the pot did not appear to influence the probability of moving for fifth instar nymphs. The difference in movement behavior of nymphs on the pots of wheat fertilized with the lowest and the highest nitrogen levels seemed to be as great as the difference in movement behavior reported earlier on two different host plant species, viz., wheat and Tripleurospermum inodorum Schultz. The magnitude of the difference in movement behavior on the extreme nitrogen levels illustrates the importance of recognizing within species variation in plant quality when relating movement patterns of insect herbivores with host plant species.     

Evaluation of the roles of two compatible solutes,glycine betaine and trehalose,for the Acacia senegal–Sinorhizobium symbiosis exposed to drought stress

Plant and Soil - Acacia senegal (Mimosoideae) is a leguminous, nitrogen-fixing tree that grows in arid areas of Africa and the Near East. In this work, we studied the effects of drought stress on...     

Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture

The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo-SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo-SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT-PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo-SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno-free component efficiently preserved CSK characteristics, with non-inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Immunolocalization of the saposin-like insert of plant aspartic proteinases exhibiting saposin C activity. Expression in young flower tissues and in barley seeds

The plant-specific insert (PSI) of cypro11 gene-encoding cyprosin, an aspartic proteinase from Cynara cardunculus , has been cloned by polymerase chain reaction (PCR) into a bacterial expression vector. A rearranged form of this PSI in which the N- and C-terminal sequences were permutated to make it more similar to the structural arrangement observed in saposins was also cloned and expressed in the same system. The biological activities of the two purified recombinant proteins were compared to those of human saposins B and C. The proteins showed similar activity to saposin C, i.e. capacity to activate human glucosylceramidase. At a concentration of 5 µ M , wild-type PSI, saposin C, and rearranged PSI activated human glucosylceramidase two-, three-, and five-fold, respectively. The K_m for 4-methylumbelliferyl β-glucopyranoside was around 7 µ M in the presence of any of the three activators (5 µ M ). The neurotropic activity using NS20Y cells and lipid-binding properties of the plant recombinant proteins were tested. The two plant proteins showed lipid-binding properties similar to those of saposins but did not have any effect on neurite outgrowth. Immunolocalization of PSI showed its expression in protective tissues in flower meristem – protodermis, in C. cardunculus and embryonic root cap and coleorhiza in mature barley grains – as well as husk, pericarp, and the aleurone layer. Possible biological functions suggested for the plant homologue to saposins besides the general activation of enzymes involved in lipid metabolism would be involvement in plant defence.     

Assembly of a filamin four-domain fragment and the influence of splicing variant-1 on the structure

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.