Identification of a Small Molecule Inhibitor of Importin β Mediated Nuclear Import by Confocal On-Bead Screening of Tagged One-Bead One-Compound Libraries

In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably,leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin beta with high nanomolar affinity and specifically inhibits importin alpha/beta mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding-experiments suggest that karyostatin 1A acts by disrupting the interaction between importin p and the OPase Ran. As a selective inhibitor of the importin alpha/beta import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.</p

Towards reconciling structure and function in the nuclear pore complex

The spatial separation between the cytoplasm and the cell nucleus necessitates the continuous exchange of macromolecular cargo across the double-membraned nuclear envelope. Being the only passageway in and out of the nucleus, the nuclear pore complex (NPC) has the principal function of regulating the high throughput of nucleocytoplasmic transport in a highly selective manner so as to maintain cellular order and function. Here, we present a retrospective review of the evidence that has led to the current understanding of both NPC structure and function. Looking towards the future, we contemplate on how various outstanding effects and nanoscopic characteristics ought to be addressed, with the goal of reconciling structure and function into a single unified picture of the NPC

Phosphorylation of nucleoporins: Signal transduction-mediated regulation of their interaction with nuclear transport receptors

The nuclear pore complex (NPC) is composed of ∼30 unique proteins, collectively referred to as nucleoporins or Nups. While metazoan Nups are known to be phosphorylated during mitosis to cause disassembly of the NPC, what is less clear is whether Nups are phosphorylated and regulated by extracellular stimuli in interphase cells. Our multi-step phosphoproteomic approach revealed a number of physiologically relevant extracellular signal-regulated kinase (ERK) targets, including Nups containing FG repeats (FG Nups) that provide binding sites for nuclear transport receptors (NTRs) during the NPC passage. The phosphorylation of FG Nups by ERK does not affect the overall architecture of the NPC but directly inhibits their interactions with NTRs and regulates the permeability barrier properties of the NPC. Such regulation at the levels of transport machinery is expected to have a broad impact on cellular physiology through the spatiotemporal control of signaling events. Until recently, many studies have focused on cellular signaling-mediated phosphorylation of individual cargo proteins, such as transcription factors. An understanding of the effects of signaling pathways on nucleocytoplasmic transport machinery is only beginning to emerge

Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex

The nuclear pore complex (NPC) is the portal for bidirectional transportation of cargos between the nucleus and the cytoplasm. While most of the structural elements of the NPC, i.e. nucleoporins (Nups), are well characterized, the exact transport mechanism is still under much debate. Many of the functional Nups are rich in phenylalanine-glycine (FG) repeats and are believed to play the key role in nucleocytoplasmic transport. We present a bioinformatics study conducted on more than a thousand FG Nups across 252 species. Our results reveal the regulatory role of polar residues and specific sequences of charged residues, named ‘like charge regions’ (LCRs), in the formation of the FG network at the center of the NPC. Positively charged LCRs prepare the environment for negatively charged cargo complexes and regulate the size of the FG network. The low number density of charged residues in these regions prevents FG domains from forming a relaxed coil structure. Our results highlight the significant role of polar interactions in FG network formation at the center of the NPC and demonstrate that the specific localization of LCRs, FG motifs, charged, and polar residues regulate the formation of the FG network at the center of the NPC