BACKGROUND AND PURPOSE: Disruption of nucleoporins has been reported in the motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS). However, the precise changes in the morphology of nucleoporins associated with the pathology of the 43-kDa TAR DNA-binding protein (TDP-43) in the disease process remain unknown. We investigated the expression of nucleoporins that constitute the nuclear pore complex (NPC) in spinal motor neurons that exhibit sALS in relation to TDP-43 pathology, which is a reliable neuropathological hallmark of sALS. METHODS: Paraffin-embedded sections of the lumbar spinal cord were obtained for immunofluorescence analysis from seven control subjects and six sALS patients. Anti-TDP-43 antibody, anti-nucleoporin p62 (NUP62) antibody, and anti-karyopherin beta 1 (KPNB1) antibody were applied as primary antibodies, and then visualized using appropriate secondary antibodies. The sections were then examined under a fluorescence microscope. RESULTS: NUP62 and KPNB1 immunoreactivity appeared as a smooth round rim bordering the nuclear margin in normal spinal motor neurons that exhibited nuclear TDP-43 immunoreactivity. sALS spinal motor neurons with apparent TDP-43 mislocalization demonstrated irregular, disrupted nuclear staining for NUP62 or KPNB1. Some atrophic sALS spinal motor neurons with TDP-43 mislocalization presented no NUP62 immunoreactivity. CONCLUSIONS: Our findings suggest a close relationship between NPC alterations and TDP-43 pathology in the degenerative process of the motor neurons of sALS patients.
Amyotrophic Lateral Sclerosis* ; Antibodies ; Fluorescence ; Fluorescent Antibody Technique ; Humans ; Motor Neurons* ; Nuclear Pore ; Nuclear Pore Complex Proteins* ; Pathology ; Spinal Cord

Transportation between the cytoplasm and the nucleoplasm is critical for many physiological and pathophysiological processes including gene expression, signal transduction, and oncogenesis. So, the molecular mechanism for the transportation needs to be studied not only to understand cell physiological processes but also to develop new diagnostic and therapeutic targets. Recent progress in the research of the nuclear transportation (import and export) via nuclear pore complex and four important factors affecting nuclear transport (nucleoporins, Ran, karyopherins, and nuclear localization signals/nuclear export signals) will be discussed. Moreover, the clinical significance of nuclear transport and its application will be reviewed. This review will provide some critical insight for the molecular design of therapeutics which need to be targeted inside the nucleus.
Active Transport, Cell Nucleus* ; Carcinogenesis ; Cell Physiological Processes ; Cytoplasm ; Gene Expression ; Karyopherins ; Nuclear Localization Signals ; Nuclear Pore ; Nuclear Pore Complex Proteins ; Signal Transduction ; Transportation

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Animals ; Artificial Intelligence ; Cryoelectron Microscopy ; Nuclear Pore/ultrastructure* ; Oocytes/metabolism* ; Xenopus laevis

Child ; Humans ; Nuclear Pore Complex Proteins/genetics* ; Leukemia, Myeloid, Acute/genetics*

Humans ; Esophageal Achalasia/genetics* ; Cardia ; Nerve Tissue Proteins ; Nuclear Pore Complex Proteins

High expression of Fightless I (FLII) is associated to multiple tumors. Based on our previous study that FLII might be involved in the nuclear export, we assessed the possible interaction of FLII with the nuclear envelop associating proteins Importin β and Nup88. We first constructed GST-FLII, GST-LRR recombinant plasmids and transformed them into the Rosetta strain to produce GST-FLII, GST-LRR fusion protein. After purification of these proteins, GST-pull down, as well as co-immunoprecipitation, were used to test the interaction of FLII with Importin β and Nup88. FLII interacted with Importin β and Nup88, and FLII LRR domain is responsible for these interactions. Thus, FLII may play a role in nuclear export through interaction with Importin β and Nup88.
Humans ; Microfilament Proteins ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Recombinant Fusion Proteins ; metabolism ; beta Karyopherins ; metabolism

In order to investigate the anti-leukemia effects of gambogic acid (GA) and its relation to the regulation of nucleoporin Nup88 in U937 cells in vitro, the inhibitory effect of GA on the growth of U937 cells was examined by using MTT assay. Apoptosis was detected by Annexin-V FITC/PI double-labeled cytometry. Cell cycle regulation was studied by propidium iodide method. Both flow cytometry (FCM) and RT-PCR were employed to assess the expression of Nup88, and the localization of Nup88 was determined by confocal microscopy. The results indicated that GA had strong inhibitory effect on cell proliferation and apoptosis induction activity in U937 cells in vitro in a time-and dose-dependent manner. The 24-h IC(50) value was (1.019+/-0.134) mg/L. Moreover, GA induced arrest of U937 cells in G(0)/G(1) phase. Over-expression of Nup88 was found in U937 cells, whereas GA could significantly down-regulate both the protein and mRNA levels of Nup88. Nup88 was diffusely distributed between nucleus and cytoplasm and was located at the cytoplasmic side of nuclear rim, and occasionally in cytoplasm. It is suggested that GA exerts its anti-leukemia effects by regulating the expression and distribution of nucleoporin Nup88. It promises to be new agent for the treatment of acute leukemia.
Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/drug effects ; Cell Proliferation/drug effects ; Nuclear Pore Complex Proteins/genetics ; Nuclear Pore Complex Proteins/*metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; U937 Cells ; Xanthones/*pharmacology

OBJECTIVETo investigate antitumor activity and molecular mechanism of deguelin to the human U937 leukaemia cells and to explore the mechanisms regulating cell cycle and nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro.

METHODSThe effects of deguelin on the growth of U937 cells were studied by MTT assay, and the cell cycle of U937 cells by a propidium iodide method. The localization of the nuclear pore complex protein Nup98 and Nup88 was checked by immunofluorescence and immunoelectron microscopy. The expressions of Nup98 and Nup88 in U937 cells were checked by flow cytometry (FCM) and Western blot respectively.

RESULTSThe proliferation of U937 cells was significantly inhibited in a time-dose dependent manner in deguelin-treated group with a 24 h IC50 value of 21.61 nmol/L and 36 h IC50 value of 17.07 nmol/L. U937 cells treated with deguelin showed reduction in the percentages of cells in G0/G1, whereas accumulation of cells in S and G2/M phase. The ratio of G1/G0 phase cells were 73.01%, 71.15%, 68.42%, 52.45%, 43.99% and 22.82%, and that of S phase cells were 17.18%, 16.30%, 18.09%, 27.56%, 31.21% and 46.85%, and that of G2/M phase cells were 9.75%, 12.31%, 13.09%, 18.99%, 24.83% and 27.79% at deguelin concentrations of 0, 5, 10, 20, 40, 80 nmol/L respectively. Nup88 and Nup98 were found on both the nuclear and cytoplasmic side of the U937 cells. The expression of Nup98 was up-regulated and Nup88 down-regulated in deguelin treated U937 cells.

CONCLUSIONDeguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle. The antitumor activity of deguelin was related to up-regulating the expression of Nup98 and down-regulating Nup88 protein.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Nuclear Pore Complex Proteins ; metabolism ; Rotenone ; analogs & derivatives ; pharmacology ; U937 Cells

Homeodomain Proteins/genetics* ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Nuclear Pore Complex Proteins ; Oncogene Proteins, Fusion/genetics* ; Translocation, Genetic

Humans ; Leukemia, Myeloid, Acute/genetics* ; Nuclear Pore Complex Proteins/genetics* ; Gene Rearrangement ; Translocation, Genetic ; Oncogene Proteins, Fusion/genetics*