Rabies, caused by lyssavirus rabies (Rabies lyssavirus, RABV), is a fatal disease among humans and almost all warm-blooded animals. In this study, we found that RABV infection induces the up-regulation of receptor transporter protein 4 (RTP4) in mouse brains and different cells of nervous tissue. Over-expression of RTP4 reduces the viral titer of RABV in different neuronal cells. Furthermore, a recombinant RABV expressing RTP4, named rRABV-RTP4, was constructed and displayed a lower viral titer in different neuronal cells due to the expression of RTP4. Moreover, the survival rates of mice infected with rRABV-RTP4 were significantly higher than those of mice infected with parent virus rRABV or control virus rRABV-RTP4(-). In terms of mechanism, RTP4 could bind viral genomic RNA (vRNA) of RABV, and suppress the whole viral genome amplification. In addition, we found that the zinc finger domain (ZFD) of RTP4 exerts the antiviral function by truncation analysis, and an important amino acids site (C95) in the RTP4 3CxxC motif which is essential for its antiviral function was identified by mutation analysis. This study contributes to our understanding of how RTP4 or other RTP proteins play a role in defense against the invasion of RABV or other viruses.

The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, male absent on the first (MOF), and maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. Here, we found that MLE\'s unstructured C-terminal region interacts with the sixth zinc-finger domain of CLAMP. In vitro, 4-5 zinc fingers are critical for the specific DNA-binding of CLAMP with GA repeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome.

Haploinsufficiency of GATA2, also known as GATA2 deficiency, leads to a wide spectrum of clinical manifestations. Here we described another 28-year-old man with a GATA2 variant who also suffered from hemophagocytic lymphohistiocytosis(HLH), who was finally diagnosed with HLH triggered by Mycobacterium avium bloodstream infection due to primary immunodeficiency. We reviewed GATA2 deficiency patients with HLH and found that GATA2 variants causing loss of zinc finger domains were associated with HLH, and erythema nodosa might be an accompanying symptom.

Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.

More than half a century ago, zinc was established as an essential micronutrient for normal human physiology. In silico data suggest that about 10% of the human proteome potentially binds zinc. Many proteins with zinc-binding domains (ZBDs) are involved in epigenetic modifications such as DNA methylation and histone modifications, which regulate transcription in physiological and pathological conditions. Zinc metalloproteins in epigenetics are mainly zinc metalloenzymes and zinc finger proteins (ZFPs), which are classified into writers, erasers, readers, editors, and feeders. Altogether, these classes of proteins engage in crosstalk that fundamentally maintains the epigenome\'s modus operandi. Changes in the expression or function of these proteins induced by zinc deficiency or loss of function mutations in their ZBDs may lead to aberrant epigenetic reprogramming, which may worsen the risk of non-communicable chronic diseases. This review attempts to address zinc\'s role and its proteins in natural epigenetic programming and artificial reprogramming and briefly discusses how the ZBDs in these proteins interact with the chromatin.

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

BACKGROUND: Epithelial-to-Mesenchymal Transition (EMT) is necessary for metastasis. Zinc- finger domain-containing transcription factors, especially Snail1, bind to E-box motifs and play a crucial role in the induction and regulation of EMT.
OBJECTIVE: We hypothesized if C-terminal region of Snail1 (CSnail1) may competitively bind to E-box and block cancer metastasis.
METHODS: The CSnail1 gene coding sequence was inserted into the pIRES2-EGFP vector. Following transfection of A549 cells with the designed construct, EMT was induced with TGF-β1 and the expression of essential EMT markers was evaluated by real-time PCR and immunoblotting. We also monitored cell migration.
RESULTS: CSnail1 inhibited TGF-β1-induced N-cadherin and vimentin mRNA expression and increased β-catenin expression in transfected TGF-β1-treated A549 cells. A similar finding was obtained in western blotting. CSnail1 also blocked the migration of transfected cells in the scratch test.
CONCLUSIONS: Transfection of A549 cells with CSnail1 alters the expression of essential EMT markers and consequently suppresses tumor cell migration. These findings confirm the capability of CSnail1 in EMT blocking and in parallel to current patents could be applied as a novel strategy in the prevention of metastasis.

GATA3 is a transcription factor, known to regulate the transcriptional network and several pathways using two zinc fingers. Its mutation is associated with a higher risk of breast cancer. The molecular mechanisms of these mutations are poorly understood. It recognizes -GATA- sites on the DNA, using its two zinc fingers ZnFn1 and ZnFn2. Mutations in ZnFn2 have been studied in the past and well known but recently ZnFn1 mutations are also being reported very frequently in breast cancer patients and there is very less knowledge available regarding the binding modes and mechanism. Here, we have investigated the structural and functional impact of GATA3 mutation M294K on the DNA-binding property. The structure was obtained and mutation was induced before subjecting it to the molecular docking followed by MD simulation. Our findings indicate that the somatic mutation M294K, reported in the GATA3 gene destabilizes the unbound structure but, when it forms the DNA-complex, its overall structural stability is restored by the wrapping architecture of ZnFn2 around the DNA in the palindromic region, leading to the enhanced kinetic stability. The mutation not only affects the ZnFn1 region alone but also influences the whole complex by inducing the conformational changes in the linker region between the two zinc fingers, bringing the two zinc fingers to closer proximity representing the flexibility in binding sites. Our findings provide a better understanding of ZnFn1 mutations and the possibility of a different strategy to target these genes for their clinical relevance.Communicated by Ramaswamy H. Sarma.

Mutations in GLI3, which encodes a transcription factor of the Hedgehog signaling pathway, cause several developmental anomalies linked to inappropriate tissue patterning. Here, we report a novel missense variant in the fifth zinc finger domain of GLI3 (c.1826G>A; p.(Cys609Tyr)) initially identified in a proband with preaxial polydactyly type IV, developmental delay, sensorineural hearing loss, skeletal, and genitourinary anomalies. Additional family members exhibited various digital anomalies such as preaxial polydactyly, syndactyly, and postaxial polydactyly either in isolation or combined. Functional studies of Cys609Tyr GLI3 in cultured cells showed abnormal GLI3 processing leading to decreased GLI3 repressor production, increased basal transcriptional activity, and submaximal GLI reporter activity with Hedgehog pathway activation, thus demonstrating an intriguing molecular mechanism for this GLI3-related phenotype. Given the complexity of GLI3 post-translational processing and opposing biological functions as a transcriptional activator and repressor, our findings highlight the importance of performing functional studies of presumed GLI3 variants. This family also demonstrates how GLI3 variants are variably expressed.