Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Flap endonuclease 1 (FEN1) is a member of the nuclease family and is structurally conserved from bacteriophages to humans. This protein is involved in multiple DNA-processing pathways, including Okazaki fragment maturation, stalled replication-fork rescue, telomere maintenance, long-patch base-excision repair and apoptotic DNA fragmentation. FEN1 has three functional motifs that are responsible for its nuclease, PCNA-interaction and nuclear localization activities, respectively. It has been shown that the C-terminal nuclear localization sequence (NLS) facilitates nuclear localization of the enzyme during the S phase of the cell cycle and in response to DNA damage. To determine the structural basis of the recognition of FEN1 by the nuclear import receptor importin alpha, the crystal structure of the complex of importin alpha with a peptide corresponding to the FEN1 NLS was solved. Structural studies confirmed the binding of the FEN1 NLS as a classical bipartite NLS; however, in contrast to the previously proposed (KRKX8KKK367)-K-354 sequence, it is the (354)KRX(10)KKAK(369) sequence that binds to importin alpha. This result explains the incomplete inhibition of localization that was observed on mutating residues (KKK367)-K-365. Acidic and polar residues in the X-10 linker region close to the basic clusters play an important role in binding to importin alpha. These results suggest that the basic residues in the N-terminal basic cluster of bipartite NLSs may play roles that are more critical than those of the many basic residues in the C-terminal basic cluster

Structural biology and regulation of protein import into the nucleus

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g. importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g. importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals (cNLSs), recognized by the adaptor importin-α, and the PY-NLSs, recognized by transportin-1. Post-translational modifications, in particular phosphorylation, constitute key regulatory mechanisms operating in these pathways

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Crystallization and preliminary X-ray crystallographic analysis of importin-alpha from Neurospora crassa

Importin-alpha recognizes cargo proteins that contain classical nuclear localization sequences (NLS) and, in complex with importin-beta, is able to translocate nuclear proteins through the nuclear pore complex. The filamentous fungus Neurospora crassa is a well studied organism that has been widely used as a model organism for fundamental aspects of eukaryotic biology, and is important for understanding the specific mechanisms of protein transport to the cell nucleus. In this work, the crystallization and preliminary X-ray diffraction analysis of importin-alpha from N. crassa (IMP alpha-Nc) complexed with a classical NLS peptide (SV40 NLS) are reported. IMP alpha-Nc-SV40 NLS crystals diffracted X-rays to 2.0 angstrom resolution and the structure was solved by molecular-replacement techniques, leading to a monomeric structure. The observation of the electron-density map indicated the presence of SV40 NLSs interacting at both the minor and major NLS-binding sites of the protein.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity already measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed

Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal

<div><p><i>Neurospora crassa</i> is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that <i>N. crassa</i> Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.</p></div

Biophysical characterization of the recombinant importin-α from neurospora crassa

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins. Copyright © 2012 Bentham Science Publishers. All Rights Reserved