Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 721-728
Author(s):  
Anya Bakhrat ◽  
Melissa S Jurica ◽  
Barry L Stoddard ◽  
Dina Raveh
Keyword(s):  
Zinc Finger ◽  
Mating Type ◽  
Mutational Analysis ◽  
Critical Role ◽  
Homing Endonucleases ◽  
Zinc Finger Domain ◽  
Catalytic Active Site ◽  
Endonuclease Domain ◽  
Putative Zinc Finger ◽  
Hint Domain

Abstract Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

scholarly journals HELZ2 Promotes K63-Linked Polyubiquitination of c-Myc to Induce Retinoblastoma Tumorigenesis

2021 ◽  
Author(s):  
Hanjun Dai ◽  
wen ZENG ◽  
WEIJUAN ZENG ◽  
MING YAN ◽  
ping jiang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Zinc Finger ◽  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Transcriptional Regulators ◽  
Zinc Finger Domain ◽  
Retinoblastoma Cell ◽  
Animal Survival

Abstract Retinoblastoma is a rare ocular tumor in children that originates in the retina. Several core transcriptional regulators maintain the expansion of retinoblastoma tumors, including c-Myc. Here, we demonstrated that Helicase with zinc finger domain 2 (HELZ2) promoted retinoblastoma tumorigenesis by targeting c-Myc. HELZ2-deficient inhibited retinoblastoma cell proliferation, whereas overexpression of HELZ2 promoted retinoblastoma cell proliferation. In addition, high levels of HELZ2 promoted xenograft retinoblastoma tumorigenesis and inhibited animal survival. Mechanistically, HELZ2 interacted with c-Myc and promoted its K63-linked polyubiquitination. We indicated that HELZ2 promoted the interaction between E3 ubiquitin ligase HUWE1 and c-Myc, and HELZ2-mediated K63-linked polyubiquitination and activation of c-Myc were dependent on HUWE1. Taken together, HELZ2 plays a critical role in the regulation of retinoblastoma tumorigenesis by enhancing the activity of c-Myc.

scholarly journals Growth factor-induced tyrosine phosphorylation of Hrs, a novel 115-kilodalton protein with a structurally conserved putative zinc finger domain.

1995 ◽  
Vol 15 (11) ◽  
pp. 6213-6221 ◽  
Author(s):  
M Komada ◽  
N Kitamura
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Zinc Finger ◽  
Amino Acid Sequences ◽  
Zinc Finger Domain ◽  
Rich Region ◽  
Putative Zinc Finger ◽  
Hepatocyte Growth

The activation of growth factor receptor tyrosine kinases leads to tyrosine phosphorylation of many intracellular proteins which are thought to play crucial roles in growth factor signaling pathways. We previously showed that tyrosine phosphorylation of a 115-kDa protein is rapidly induced in cells treated with hepatocyte growth factor. To clarify the structure and possible function of the 115-kDa protein (designated Hrs for hepatocyte growth factor-regulated tyrosine kinase substrate), we purified this protein from B16-F1 mouse melanoma cells by anti-phosphotyrosine immunoaffinity chromatography and determined its partial amino acid sequences. On the basis of the amino acid sequences, we molecularly cloned the cDNA for mouse Hrs. The nucleotide sequence of the cDNA revealed that Hrs is a novel 775-amino-acid protein with a putative zinc finger domain that is structurally conserved in several other proteins. This protein also contained a proline-rich region and a proline- and glutamine-rich region. The expression of Hrs mRNA was detected in all adult mouse tissues tested and also in embryos. To analyze the Hrs cDNA product, we prepared a polyclonal antibody against bacterially expressed Hrs. Using this antibody, we showed by subcellular fractionation that Hrs is localized to the cytoplasm; we also showed that that tyrosine phosphorylation of Hrs is induced in cells treated with epidermal growth factor or platelet-derived growth factor. These results suggest that Hrs plays a unique and important role in the signaling pathway of growth factors.

scholarly journals Mutations in Influenza Virus M1 CCHH, the Putative Zinc Finger Motif, Cause Attenuation in Mice and Protect Mice against Lethal Influenza Virus Infection

2006 ◽  
Vol 80 (12) ◽  
pp. 5697-5707 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Donald F. Smee ◽  
Min-Hui Wong ◽  
Debi P. Nayak
Keyword(s):  
Influenza Virus ◽  
Zinc Finger ◽  
Cultured Cells ◽  
Critical Role ◽  
Influenza Virus Infection ◽  
Growth Patterns ◽  
Yield Potential ◽  
Zinc Finger Motif ◽  
Cells In Culture ◽  
Putative Zinc Finger

ABSTRACT Mutations in CCHH, the putative zinc finger motif, apparently do not play an important role in virus replication in MDCK cells in culture (E. K.-W. Hui, K. Ralston, A. K. Judd, and D. P. Nayak, J. Gen. Virol. 84:3105-3113, 2003). In this report, however, we demonstrate that the CCHH motif plays a critical role in virulence in mice and that some CCHH mutants are highly attenuated in BALB/c mice. Some of the mutant viruses replicated the least in mice lungs, induced little or no lung lesions, and caused highly reduced morbidity and mortality. Furthermore, growth patterns of mutant viruses in different cell lines (MDCK, MLE12, 3LL, A549, and 293T) varied. Mutant viruses that were attenuated in mice also grew poorly in mouse and human cells in culture. However, wild-type (WT) and all mutant viruses replicated to the same titer in MDCK (canine) cells or embryonated chicken eggs. Attenuation in mice correlated with reduced growth in mouse cells in culture, suggesting that potential attenuation in a given host can be predicted from the growth characteristics of the virus in cultured cells (preferably lung cells) from the same species. In challenge experiments, mice immunized by infection with attenuated mutant viruses were fully protected from lethal challenge with WT virus. In summary, the replication and attenuating properties of these mutants suggest that the CCHH motif provides a critical determinant for virulence in mouse and that mutations in the CCHH motif yield potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines.

scholarly journals The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nikolay Dobrev ◽  
Yasar Luqman Ahmed ◽  
Anusree Sivadas ◽  
Komal Soni ◽  
Tamás Fischer ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Mutational Analysis ◽  
Zinc Finger Domain ◽  
Yeast Two Hybrid ◽  
Finger Protein ◽  
Nuclear Exosome ◽  
Interaction Map ◽  
Two Hybrid

AbstractCryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Hsp70 Escort Protein: More Than a Regulator of Mitochondrial Hsp70

2018 ◽  
Vol 16 (1) ◽  
pp. 64-73 ◽  
Author(s):  
David O. Nyakundi ◽  
Stephen J. Bentley ◽  
Aileen Boshoff
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Domain ◽  
C Terminus ◽  
Cellular Localisation ◽  
The Matrix ◽  
Mitochondrial Hsp70 ◽  
Cellular Compartments ◽  
Intramolecular Communication ◽  
Self Aggregation ◽  
J Protein

Hsp70 members occupy a central role in proteostasis and are found in different eukaryotic cellular compartments. The mitochondrial Hsp70/J-protein machinery performs multiple functions vital for the proper functioning of the mitochondria, including forming part of the import motor that transports proteins from the cytosol into the matrix and inner membrane, and subsequently folds these proteins in the mitochondria. However, unlike other Hsp70s, mitochondrial Hsp70 (mtHsp70) has the propensity to self-aggregate, accumulating as insoluble aggregates. The self-aggregation of mtHsp70 is caused by both interdomain and intramolecular communication within the ATPase and linker domains. Since mtHsp70 is unable to fold itself into an active conformation, it requires an Hsp70 escort protein (Hep) to both inhibit self-aggregation and promote the correct folding. Hep1 orthologues are present in the mitochondria of many eukaryotic cells but are absent in prokaryotes. Hep1 proteins are relatively small and contain a highly conserved zinc-finger domain with one tetracysteine motif that is essential for binding zinc ions and maintaining the function and solubility of the protein. The zinc-finger domain lies towards the C-terminus of Hep1 proteins, with very little conservation outside of this domain. Other than maintaining mtHsp70 in a functional state, Hep1 proteins play a variety of other roles in the cell and have been proposed to function as both chaperones and co-chaperones. The cellular localisation and some of the functions are often speculative and are not common to all Hep1 proteins analysed to date.

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