Role of karyopherin nuclear transport receptors in nuclear transport by nuclear trafficking peptide.

Nuclear trafficking peptide (NTP), a cell-penetrating peptide (CPP) composed of 10 amino acids (aa) (RIFIHFRIGC), has potent nuclear trafficking activity. Recently, we established a protein-based cell engineering system by using NTP, but it remained elusive how NTP functions as a CPP with nuclear orientation. In the present study, we identified importin subunit ß1 (IMB1) and transportin 1 (TNPO1) as cellular proteins underlying the activity of NTP. These karyopherin nuclear transport receptors were identified as candidate molecules by liquid chromatography/mass spectrometry analysis, and downregulation of each protein by small interfering RNA significantly reduced NTP activity (P < 0.01). Biochemical analyses revealed that NTP bound directly to both molecules, and the forced expression of an IMB1 fragment (296-516 aa) or TNPO1 fragment (1-297 aa), which both contain binding sites to NTP, reduced nuclear NTP-green fluorescent protein (GFP) levels when it was added to cell culture medium. NTP is derived from viral protein R (Vpr) of human immunodeficiency virus-1, and Vpr enters the nucleus and exerts pleiotropic functions. Notably, Vpr bound directly to IMB1 and TNPO1, and its function was significantly impaired by the forced expression of the 296-516-aa fragment of IMB1 and 1-297-aa fragment of TNPO1. Interestingly, NTP completely blocked the physical association of Vpr with IMB1 and TNPO1. Although the nuclear localization mechanism of Vpr remains unknown, our data suggest that NTP functions as a novel nuclear localization signal of Vpr.

Retrotransposition and senescence in mouse heart tissue by viral protein R of human immunodeficiency virus-1.

Combination antiretroviral therapy (cART) has greatly improved the prognosis of patients with human immunodeficiency virus type-1 (HIV-1) infection. However, cardiovascular disease (CVD) remains a serious issue even in the post-cART era. Viral protein R (Vpr), an accessory gene product of HIV-1, exerts pleiotropic activities such as the induction of DNA damage signals, apoptosis by mitochondrial membrane depolarization, G2/M-phase cell cycle abnormalities, and retrotransposition. Importantly, some of these cellular responses are induced by the trans-acting activity of Vpr. Recently, we established an enzyme-linked immunosorbent assay to detect Vpr and reported that about 22% of blood samples from 100 HIV-1-positive patients were positive for Vpr. Here, we investigated the biological effects of recombinant Vpr (rVpr) in vivo. We observed that repeated injections of rVpr increased the copy number of long interspersed element-1 (L1) in the heart genome in mice. rVpr also increased the number of cells positive for senescence-associated ß-galactosidase (SA-ß-gal) and fibrosis in the heart. Notably, co-administration of a reverse transcriptase inhibitor reduced the number of rVpr-induced SA-ß-gal-positive cells and fibrosis concomitantly with the attenuation of L1 retrotransposition. Interestingly, a Vpr mutant defective for mitochondrial dysfunction also induced heart senescence and increased L1 copy number. Together with a recent report that L1 retrotransposition functions as a molecular basis of senescence, our current data suggest that rVpr-induced L1 retrotransposition is linked with senescence in heart tissue. We would propose that Vpr in the bloodstream may be one of risk factors for CVD, and that its monitoring will lead to well understanding of the heterogeneity and multifactorial mechanisms of CVD in HIV-1 patients.

Development of a protein-based system for transient epigenetic repression of immune checkpoint molecule and enhancement of antitumour activity of natural killer cells.

BACKGROUND: Immune checkpoint blockade (ICB) therapy improved the prognosis of cancer patients, but general administration of ICBs occasionally induces side effects that include immune-related adverse events and tumour hyper-progression. Here, we established a protein-based system, by which endogenous expression of IC molecule in natural killer (NK) cells was transiently repressed on enhancement of their antitumour activity. METHODS: A protein-based genome modulator (GM) system is composed of a transcription activator-like effector (TALE), DNA methyltransferase and a newly identified potent cell-penetrating peptide with nuclear-trafficking property named NTP. TALE was designed to target the promoter region of the programmed cell death-1 (PD-1) gene. After culturing human NK cells in the presence of NTP-GM protein, we examined endogenous PD-1 expression and antitumour activity of the treated cells. RESULTS: NTP-GM protein efficiently downregulated PD-1 expression in NK cells with increased CpG DNA methylation in the promoter region. The antitumour activity of the treated NK cells was enhanced, and repeated intraperitoneal administrations of the treated NK cells attenuated tumour growth of programmed death-ligand 1-positive tumour cells in vivo. CONCLUSIONS: Because the incorporated NTP-GM protein was quickly degraded and negligible in the administered NK cells, the NTP-GM system could be an alternative option of an ICB without side effects.