Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus

Influence of zinc sulfate intake on acute ethanol-induced liver injury in rats

AIM: To investigate the role of metallothionein and proliferating cell nuclear antigen (PCNA) on the morphological and biochemical effects of zinc sulfate in ethanol-induced liver injury. METHODS: Wistar albino rats were divided into four groups. Group I; intact rats, group II; control rats given only zinc, group III; animals given absolute ethanol, group IV; rats given zinc and absolute ethanol. Ethanol-induced injury was produced by the 1 mL of absolute ethanol, administrated by gavage technique to each rat. Animals received 100 mg/kg per day zinc sulfate for 3 d 2 h prior to the administration of absolute ethanol. RESULTS: Increases in metallothionein immunoreactivity in control rats given only zinc and rats given zinc and ethanol were observed. PCNA immunohistochemistry showed that the number of PCNA-positive hepatocytes was increased significantly in the livers of rats administered ethanol + zinc sulfate. Acute ethanol exposure caused degenerative morphological changes in the liver. Blood glutathione levels decreased, serum alkaline phosphatase and aspartate transaminase activities increased in the ethanol group when compared to the control group. Liver glutathione levels were reduced, but lipid peroxidation increased in the livers of the group administered ethanol as compared to the other groups. Administration of zinc sulfate in the ethanol group caused a significant decrease in degenerative changes, lipid peroxidation, and alkaline phosphatase and aspartate transaminase activities, but an increase in liver glutathione. CONCLUSION: Zinc sulfate has a protective effect on ethanol-induced liver injury. In addition, cell proliferation may be related to the increase in metallothionein immunoreactivity in the livers of rats administered ethanol + zinc sulfate

Influence of zinc sulfate intake on acute ethanol-induced liver injury in rats

AIM: To investigate the role of metallothionein and proliferating cell nuclear antigen (PCNA) on the morphological and biochemical effects of zinc sulfate in ethanol-induced liver injury

Alterations in Somatostatin Cells and Biochemical Parameters Following Zinc Supplementation in Gastrointestinal Tissue of Streptozotocin-Induced Diabetic Rats

Chronic hyperglycemia in diabetes is a major causative factor of free radical generation which further leads to many secondary diabetic complications via the damage to cellular proteins, membrane lipids, and nucleic acids. Zinc is an essential trace element in all living systems and plays a structural role in many proteins and enzymes. Somatostatin is known to have inhibitory effects on various gastrointestinal functions. Therefore, we determined somatostatin protein production and secretion levels, and biochemical and light microscopical changes following zinc supplementation in the gastrointestinal tract of streptozotocin (STZ)-diabetic rats. The animals were divided into four groups: Group I: control (untreated) animals; Group II: control animals given zinc sulfate; Group III: diabetic animals; and Group IV: diabetic animals given zinc sulfate. Zinc sulfate was given to the animals by gavage at a daily dose of 100 mg/kg body weight for 60 days. Diabetes was induced by intraperitoneal (i.p.) injection of STZ in a single dose of 65 mg/kg. For histological studies, stomach and duodenum tissues were fixed in Bouin solution and sections stained with Masson’s trichrome and Periodic-Acid-Schiff. Tissue homogenates were used for protein, lipid peroxidation (LPO), glutathione (GSH), and nonenzymatic glycosylation (NEG) analyses. Zinc supplementation to the STZ-diabetic rats revealed the protective effect of zinc on these parameters. Zinc supplementation may contribute to prevent at least some complications of diabetes mellitus

Nuclear Phosphoinositides—Versatile Regulators of Genome Functions

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), modulate nuclear processes. We focus on PI(4,5)P2’s role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets—recently described nucleoplasmic PI(4,5)P2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide–protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies

Neck domain of NM1 contains the NLS.

<p>U2OS cell transfected with a panel of truncation constructs of full length NM1 (<b>A–F</b>) and IQ domains fused to GFP-PK (<b>G–L</b>). Cells were fixed 48 hours post transfection. Below the pictures are schematic representations of the truncations affecting various NM1 domains as well as the GFP-PK phusions. Pictures (<b>A–F</b>) were acquired using confocal microscope, single confocal planes are shown. Pictures (<b>G–L</b>) were photographed using wide-field fluorescent microscope. Scale bar: 10 µm.</p

Overexpression of calmodulin influences the nuclear import of NM1.

<p>U2OS cells were co-transfected with GFP-PK constructs containing IQ domains, and calmodulin. Calmodulin was visualized using specific antibody (<b>A</b>,<b>B</b>,<b>C</b>). Scale bar 10 µm. HEK293T cells electroporated with the same constructs as in (<b>A</b>,<b>B</b>,<b>C)</b>. Whole cell extracts were subjected to immunoprecipitation with anti-GFP nanobody. Bound proteins were resolved on SDS-PAGE and transferred to nitrocelulose. GFP and CaM were visualized using specific antibodies (<b>D</b>). (<b>E</b>) Comparison of IQ1 and IQ2 sequences. The consensus IQ motif is shown below. The NM1 NLS sequence is highlighted in red.</p