Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA.

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis

Protein composition of the subretinal fluid suggests selective diffusion of vitreous proteins in retinal detachment

Purpose: To study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD. Methods: Human undiluted SRF obtained during vitrectomy for primary RRD using a 41-gauge needle (n = 24) was analyzed and compared to vitreous humor from 2-day postmortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography–tandem mass spectrometry. Label-free quantification (LFQ) using MaxQuant was used to determine differentially expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine. Results: The protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 38 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ, and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF. Conclusions: The proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD. Translational Relevance: To identify potential novel biomarkers for therapeutic targeting in RD

The low-density lipoprotein receptor-related protein 1 (LRP1) interactome in the human cornea

The human cornea is responsible for approximately 70% of the eye's optical power and, together with the lens, constitutes the only transparent tissue in the human body. Low-density lipoprotein receptor-related protein 1 (LRP1), a large, multitalented endocytic receptor, is expressed throughout the human cornea, yet its role in the cornea remains unknown. More than 30 years ago, LRP1 was purified by exploiting its affinity for the activated form of the protease inhibitor alpha-2-macroblulin (A2M), and the original purification protocol is generally referred to in studies involving full-length LRP1. Here, we provide a novel and simplified LRP1 purification protocol based on LRP1's affinity for receptor-related protein (RAP) that produces significantly higher yields of authentic LRP1. Purified LRP1 was used to map its unknown interactome in the human cornea. Corneal proteins extracted under physiologically relevant conditions were subjected to LRP1 affinity pull-down, and LRP1 ligand candidates were identified by LC-MS/MS. A total of 28 LRP1 ligand candidates were found, including 22 novel ligands. The LRP1 corneal interactome suggests a novel role for LRP1 as a regulator of the corneal immune response, structure, and ultimately corneal transparency

Mutation-induced dimerization of transforming growth factor-β–induced protein may drive protein aggregation in granular corneal dystrophy

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β–induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography–small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

<div><p>Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.</p></div

Aldosterone administration increases proteasome numbers and labeling intensity in distal renal tubules.

<p>A) Double labeling for proteasomes (proteasome 20 s, green) and a marker for DCT (NCC, red) in renal cortex from control rats. B) Similar fluorescence labeling in renal cortex from aldosterone treated rats. C) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in DCT (Con and Aldo, as indicated, n = 5). D) Double labeling of proteasomes (proteasome 20 s, red) and a marker for CNT (calbindin-D_28K, blue) in renal cortex from control rats. E) Similar fluorescence labeling in renal cortex from aldosterone treated rats. F) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in CNT (Con and Aldo, as indicated, n = 5). * indicates statistical significance.</p

Co-localization of proteasome 20 s aggregates and HDAC6 after aldosterone administration.

<p>A) Double fluorescence labeling for RPL22 aggregates (H-95, red) and HDAC6 (green) in kidney from control rat. B) Similar labeling in kidney from aldosterone treated rat. Arrow heads indicate colocalization (in yellow). C) The immunostaing for the de-ubiquitinase ataxin-3 in control rat kidney sections. D) Representative micrograph of renal cortex of an aldosterone treated rat stained for ataxin-3. E) Double fluorescence labeling for aggresome initiator protein p62 (red) and HDAC6 (green) in kidney from control rat. F) Similar labeling in kidney from aldosterone treated rat. G) Renal cortex from control rats was labeled for autophagosomes by anti-LC3 antibodies (green) and overlaid on DIC image. H) Similar fluorescence labeling of renal cortex from aldosterone treated rat. I) Similar staining where distal renal tubules were identified by calbindin-D_28K (red). J) Corresponding micrograph from aldosterone treated rat. “PT” marks proximal tubules while “DT” are distal renal tubules and connecting tubules.</p