A theory on SARS-COV-2 susceptibility: reduced TLR7-activity as a mechanistic link between men, obese and elderly.

I hypothesize that chronic stimulation of tlr7 by intrinsic substrates in old and obese people leads to a desensitization of tlr7-signalling such that upon viral infection the immune response in those patients will be delayed, allowing the virus to more easily establish itself in these at-risk patient groups. Upon severe viral infection, resensitization of tlr7-signalling occurs so as to better fight the viral infection. This, however, leads to a situation where old and obese patients develop an overwhelming tlr7-response due to the presence of not only the viral RNA, but also the intrinsic tlr7 substrates. Thus, old and obese patients with serious COVID-19 develop a state that is similar to SLE-patients and consequently should be treated in a similar manner. The theory predicts that drugs and treatment regimens that help to control SLE-flares might also help in late-stage COVID-19 patients.

Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis.

BACKGROUND: The transport of macromolecules between the nucleus and cytoplasm is an energy-dependent process. Substrates are translocated across the nuclear envelope through nuclear pore complexes (NPCs). Translocation requires nucleocytoplasmic transport receptors of the importin beta family, which interact both with the NPC and, either directly or via an adaptor, with the transport substrate. Although certain receptors have recently been shown to cross the NPC in an energy-independent manner, translocation of substrate-receptor complexes through the NPC has generally been regarded as an energy-requiring step. RESULTS: We describe an in vitro system that is based on permeabilised cells and supports nuclear export mediated by leucine-rich nuclear export signals. In this system, export is dependent on exogenous CRM1/Exportin1 - a nuclear export receptor - the GTPase Ran and nucleotide triphosphates (NTPs), and is further stimulated by Ran-binding protein 1 (RanBP1) and nuclear transport factor 2 (NTF2). Unexpectedly, non-hydrolysable NTP analogues completely satisfy the NTP requirements for a single-round of CRM1-mediated translocation of protein substrates across the NPC. Similarly, single transportin-mediated nuclear protein import events are shown not to require hydrolysable NTPs and to occur in the absence of the Ran GTPase. CONCLUSIONS: Our data show that, contrary to expectation and prior conclusions, the translocation of substrate-receptor complexes across the NPC in either direction occurs in the absence of NTP hydrolysis and is thus energy independent. The energy needed to drive substrate transport against a concentration gradient is supplied at the step of receptor recycling in the cytoplasm.

Nucleocytoplasmic transport: the soluble phase.

Active transport between the nucleus and cytoplasm involves primarily three classes of macromolecules: substrates, adaptors, and receptors. Some transport substrates bind directly to an import or an export receptor while others require one or more adaptors to mediate formation of a receptor-substrate complex. Once assembled, these transport complexes are transferred in one direction across the nuclear envelope through aqueous channels that are part of the nuclear pore complexes (NPCs). Dissociation of the transport complex must then take place, and both adaptors and receptors must be recycled through the NPC to allow another round of transport to occur. Directionality of either import or export therefore depends on association between a substrate and its receptor on one side of the nuclear envelope and dissociation on the other. The Ran GTPase is critical in generating this asymmetry. Regulation of nucleocytoplasmic transport generally involves specific inhibition of the formation of a transport complex; however, more global forms of regulation also occur.

Energy- and temperature-dependent in vitro export of RNA from synthetic nuclei.

We describe a novel assay for the study of RNA export from the nucleus in vitro. Nuclei are assembled in Xenopus egg extract on paramagnetic beads coated with DNA containing a specific template for transcription. T7 RNA polymerase, to which a nuclear localisation signal is attached, is added to the nuclei, and after its import into the assembled nuclei, transcription is allowed to proceed. The use of radioactive NTPs coupled with the possibility to purify the nuclei on a magnet and thus rapidly change the extract in which the nuclei are incubated allows pulse-chase labelling experiments. Using these protocols we show that U1 snRNA-derived templates are transcribed inside the synthetic nuclei, and that the transcripts leave the intact nuclei in a time-, temperature- and energy-dependent way. This offers the possibility of a biochemical approach to the dissection of RNA export.

RanBP3 influences interactions between CRM1 and its nuclear protein export substrates.

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1-export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.

The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP.

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.