Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades’ response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol–gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Complexation of 26-Mer Amylose with Egg Yolk Lipids with Different Numbers of Tails Using a Molecular Dynamics Simulation

A molecular dynamics simulation of mixtures of 26-mer amylose with three different egg yolk lipids, namely, cholesterol, triglyceride and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), demonstrated the formation of a stable complex. The 26-mer amylose fluctuated between a coiled and an extended helical conformation. The complex was a V-type amylose complex, with the hydrophobic tail of the lipids being inside the hydrophobic helical cavity of the amylose. The number of glucose units per turn was six for the two helical regions of the amylose-POPC complex and the palmitoyl tail region of the amylose-triglyceride complex. This value was eight for the cholesterol and the two-tail helical region in the amylose-triglyceride complex. Two tails of the POPC were in two different hydrophobic helical regions of the 26-mer amylose, whereas the palmitoyl tail of the triglyceride lay in one hydrophobic helical region and the linoleoyl and oleoyl tails both lay in another helical region, and the cross-sectional area of the latter was larger than the former to accommodate the two tails. The radii of the gyration of the complex were lower for all three cases compared to that of one single amylose. In addition, the stability of the complexes was ranked in the following order: POPC < cholesterol < triglyceride, with their average binding energy being −97.83, −134.09, and −198.35 kJ/mol, respectively.

Mutant collagen COL11A1 enhances cancerous invasion

Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of β1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade

Anhydrobiosis is one of the most extensively studied forms of cryptobiosis that is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. However, the molecular mechanisms underlying these protein functions remain to be fully elucidated. In this study, in vitro and in vivo characterizations of the self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. Our in vitro observations showed that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased, and that these molecular assembling processes were reversible. Furthermore, our in vivo observations demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into the protective mechanisms involved in the anhydrobiosis of tardigrades.

Structural analyses of an RNA stability element interacting with poly(A)

Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (∼24 kDa and ∼33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo‐electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENE-poly(A) interaction involves a motif that protects the 3ʹ-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)–RNA interaction opens new venues for better understanding of poly(A) tail biology.

Membrane insertion of Cavin1 facilitates caveolae assembly

Caveolae are small cell surface invaginations, important for control of membrane tension, signaling cascades and lipid sorting. Their formation is coupled to the lipid-dependent oligomerization of the proteins Caveolin1 and Cavin1, which are essential for membrane curvature generation. Yet, the mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical techniques to show that Cavin1 inserts into membranes. We found that the helical region 1 (HR1) initiated membrane binding via electrostatic interactions, which is further enforced by partial helical insertion in a PI(4,5)P2-dependent process mediated by the disordered region 1 (DR1). In agreement with this, the DR1 was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that DR1 and HR1 of Cavin1 constitute a novel membrane interacting unit facilitating dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Exon-Trapping Assay Improves Clinical Interpretation of COL11A1 and COL11A2 Intronic Variants in Stickler Syndrome Type 2 and Otospondylomegaepiphyseal Dysplasia

Stickler syndrome (SS) is a hereditary connective tissue disorder affecting bones, eyes, and hearing. Type 2 SS and the SS variant otospondylomegaepiphyseal dysplasia (OSMED) are caused by deleterious variants in COL11A1 and COL11A2, respectively. In both genes, available database information indicates a high rate of potentially deleterious intronic variants, but published evidence of their biological effect is usually insufficient for a definite clinical interpretation. We report four previously unpublished intronic variants in COL11A1 (c.2241 + 5G>T, c.2809 − 2A>G, c.3168 + 5G>C) and COL11A2 (c.4392 + 1G>A) identified in type 2 SS/OSMED individuals. The pathogenic effect of these variants was first predicted in silico and then investigated by an exon-trapping assay. We demonstrated that all variants can induce exon in-frame deletions, which lead to the synthesis of shorter collagen XI α1 or 2 chains. Lacking residues are located in the α-triple helical region, which has a crucial role in regulating collagen fibrillogenesis. In conclusion, this study suggests that these alternative COL11A1 and COL11A2 transcripts might result in aberrant triple helix collagen. Our approach may help to improve the diagnostic molecular pathway of COL11-related disorders.

Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade

ABSTRACT Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members. IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

Characterisation of a Novel A-Superfamily Conotoxin

Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins.