Hath domain of hepatoma-derived growth factor (hHDGF)

The PWWP domain of mammalian DNA methyltransferase

The PWWP domain from S. pombe protein SPBC215.07c

Sequence specific 1H, 13C and 15N resonance assignments of the hath-domain of human hepatoma-derived growth factor.

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1 ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1 microg/ml. The expression of the fusion proteins, bHRP-4-green fluorescent protein and mHDGF-green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42 nM. Comparison of the bHRP-4 amino acid sequence with HRP-1-3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.
Dietz F, Franken S, Yoshida K, Nakamura H, Kappler J, Gieselmann V. The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies. Biochem J. 2002 Sep 1;366(Pt 2):491-500.
The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies.

Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.

Kishima Y, Yamamoto H, Izumoto Y, Yoshida K, Enomoto H, Yamamoto M, Kuroda T,
Ito H, Yoshizaki K, Nakamura H.
Hepatoma-derived growth factor stimulates cell growth after translocation to
the nucleus by nuclear localization signals.
J Biol Chem. 2002 Mar 22;277(12):10315-22. Epub 2001 Dec 18.

Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals.

The PWWP domain is a ubiquitous eukaryotic protein module characterised by a region of sequence similarity of approximately 80 amino acids containing a highly conserved PWWP motif. It is frequently found in proteins associated with chromatin. We have determined the structure of a PWWP domain from the S. pombe protein SPBC215.07c using NMR spectroscopy. The structure is composed of a five stranded beta barrel followed by two alpha helices. Comparison to the recently reported structure of a homologous domain from the mammalian DNA methyltransferase Dnmt3b reveals substantial differences both in the C-terminal helical region and in the PWWP motif.

Slater LM, Allen MD, Bycroft M.
Structural variation in PWWP domains.
J Mol Biol. 2003 Jul 11;330(3):571-6.

We recently identified hepatoma-derived growth factor (HDGF) as a nuclear targeted vascular smooth muscle cell (VSM) mitogen that is expressed in developing vascular lesions. In the present study, VSM in culture express endogenous HDGF only in the nucleus and target a green fluorescent protein (GFP)-HDGF fusion to the nucleus. To define the features of the HDGF molecule that are essential for nuclear localization and mitogenic function, deletion and site-directed mutagenesis were performed. Deletion analysis identified the carboxyl-terminal half of HDGF to be responsible for nuclear targeting in VSM. Overexpression of tagged HDGF proteins with point mutations in the putative bipartite nuclear localization sequence in the carboxyl terminus demonstrated that single Lys --> Asn mutations randomized HDGF expression to both the nucleus and cytoplasm similar to the empty vector. Importantly, the Lys --> Asn mutation of all three lysines blocked nuclear entry. Point mutation of a p34(cdc2) kinase consensus motif within the nuclear localization sequence had no effect on nuclear targeting. Moreover, nuclear entry was essential for the HDGF mitogenic effect, as transfection with the triple Lys --> Asn mutant HA-HDGF significantly attenuated bromodeoxyuridine uptake when compared with transfection with wild type HA-HDGF. We conclude that HDGF contains a true bipartite nuclear localization sequence with all three lysines necessary for nuclear targeting. Nuclear targeting of HDGF is required for HDGF stimulation of DNA replication in VSM.

Everett AD, Stoops T, McNamara CA.
Nuclear targeting is required for hepatoma-derived growth factor-stimulated
mitogenesis in vascular smooth muscle cells.
J Biol Chem. 2001 Oct 5;276(40):37564-8. Epub 2001 Jul 31.

Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells.

The protein contains N-terminal 10 residue His-tag, MHHHHHHAMA. Hath domain also contains a PWWP domain (residue 12 to 68), which might play a role in protein-protein interaction.

Nmr Structure Of The N-Terminal Hath Domain Of Human Hdgf

High Resolution Structure Of The Hdgf Pwwp Domain

Domain-Swapped Dimer Of The Pwwp Module Of Human Hepatoma- Derived Growth Factor

hepatoma derived growth factor [Mus musculus domesticus]

hepatoma derived growth factor [Bos taurus]

Hepatoma-derived growth factor [Mus musculus]

Hepatoma-derived growth factor [Rattus norvegicus]

hepatoma-derived growth factor isoform b [Homo sapiens]

hepatoma-derived growth factor isoform c [Homo sapiens]

hepatoma-derived growth factor isoform a [Homo sapiens]

RecName: Full=Hepatoma-derived growth factor; Short=HDGF; AltName: Full=High mobility group protein 1-like 2; Short=HMG-1L2 [Ho

RecName: Full=Hepatoma-derived growth factor; Short=HDGF [Mus musculus]

RecName: Full=Hepatoma-derived growth factor; Short=HDGF [Rattus norvegicus]

RecName: Full=Hepatoma-derived growth factor; Short=HDGF [Bos taurus]

TPA: hepatoma-derived growth factor [Bos taurus]

E. coli system was used for protein expression.

Huama Hepatoma-derived growth factor (hHDGF)

1H, 13C, and 15N Resonance Assignments of the Hath-Domain of Hepatoma-derived Growth Factor

NMR spectrometers were equipped with TXI quadruple probe.