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

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)

NMR spectrometers were equipped with TXI quadruple probe.