Molecular structure of human geminin.
ABSTRACT The origin licensing repressor geminin is a unique bifunctional protein providing a molecular link between cellular proliferation, differentiation and genomic stability. Here we report the first molecular structure of human geminin, determined by EM and image processing at a resolution of 17.5 A. The geminin molecule is a tetramer formed by two dimers with monomers interacting via coiled-coil domains. The unusual structural organization of geminin provides molecular insight into its bifunctional nature.
- SourceAvailable from: David L Selwood[Show abstract] [Hide abstract]
ABSTRACT: In order to deliver siRNA for therapeutic use several hurdles must be addressed. Metabolic degradation must be blocked, the RNAi cellular machinery is located in the cytoplasm, while double stranded siRNA is large, highly charged and impermeable to cell membranes. To date the solutions to the delivery issues have mostly involved different forms of lipid particle encapsulation. Cell penetrating peptides (CPPs) and their mimics or analogues offer a different approach and this is an emerging field with the first in vivo examples now reported. Recent reports point to lipid receptors being involved in the cellular uptake of both types of transporter. This review examines the delivery of siRNA with a focus on CPPs and their small molecule and oligomeric mimics. The current status of siRNA delivery methods in clinical trials is examined. It now seems that the goal of delivering siRNA therapeutically is achievable but will they form part of a sustainable healthcare portfolio for the future. © 2012 John Wiley & Sons A/S.Chemical Biology & Drug Design 09/2012; · 2.47 Impact Factor
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ABSTRACT: Balloon cells (BCs) are the pathologic hallmark of focal cortical dysplasia type IIB, a common cause of pharmacoresistent epilepsy. Expression of markers of cell immaturity and of the proliferation marker minichromosome maintenance protein 2 (mcm2) have been previously shown in BCs, suggesting that these cells might represent a pool of less-differentiated cells licensed for replication. An alternative explanation is that these cells are the remnants of early cortical plate cells that have failed to differentiate or to be eliminated during development and are arrested in the cell cycle, a hypothesis that this study aims to explore. Using immunohistochemical methods and semiquantitative analysis in 19 cases of focal cortical dysplasia (ages 1-81 years), we studied the expression of cell cycle proteins important either in regulating progression through the G1 phase or inducing cell arrest and promoting premature senescence. Only a small fraction of BCs expressed geminin, suggesting that few BCs enter the S phase or complete the cell cycle. Variable expression of nonphosphorylated retinoblastoma protein (Rb), cdk4, and p53 was noted in BCs. Cyclin E, D1, cdk2, phosphorylated Rb (795 and 807/811), and checkpoint 2 expression levels were low in BCs. These findings suggest early rather than late G1 arrest. Cell senescence could be induced by an undefined cerebral insult during development or alternatively represent a physiologic replicative senescence. These findings also suggest that dysregulation of cell cycle pathways may occur in focal cortical dysplasia, which opens further areas for exploration as potential new treatment avenues.Journal of Neuropathology and Experimental Neurology 01/2007; 66(11):1045-1055. · 4.35 Impact Factor
Article: The cell cycle and cancer.[Show abstract] [Hide abstract]
ABSTRACT: Deregulation of the cell cycle underlies the aberrant cell proliferation that characterizes cancer and loss of cell cycle checkpoint control promotes genetic instability. During the past two decades, cancer genetics has shown that hyperactivating mutations in growth signalling networks, coupled to loss of function of tumour suppressor proteins, drives oncogenic proliferation. Gene expression profiling of these complex and redundant mitogenic pathways to identify prognostic and predictive signatures and their therapeutic targeting has, however, proved challenging. The cell cycle machinery, which acts as an integration point for information transduced through upstream signalling networks, represents an alternative target for diagnostic and therapeutic interventions. Analysis of the DNA replication initiation machinery and mitotic engine proteins in human tissues is now leading to the identification of novel biomarkers for cancer detection and prognostication, and is providing target validation for cell cycle-directed therapies.The Journal of Pathology 01/2012; 226(2):352-64. · 7.59 Impact Factor
Molecular structure of human
Andrei L Okorokov1,3–5, Elena V Orlova2,5, Sarah R Kingsbury3,
Claire Bagneris1, Ulrich Gohlke1, Gareth H Williams3,4&
The origin licensing repressor geminin is a unique bifunctional
protein providing a molecular link between cellular
proliferation, differentiation and genomic stability. Here
we report the first molecular structure of human geminin,
determined by EM and image processing at a resolution of
17.5 Å. The geminin molecule is a tetramer formed by two
dimers with monomers interacting via coiled-coil domains.
The unusual structural organization of geminin provides
molecular insight into its bifunctional nature.
Initiation of eukaryotic chromosomal replication is dependent on the
sequential assembly of ORC, Cdc6, Cdt1 and MCM proteins into pre-
replicative complexes (pre-RCs), thus rendering origins licensed for
one round of DNA replication during S phase1. Replication initiation
is tightly coupled to removal of the license and thus prevention of reli-
censing after origin firing. Metazoan cells have adopted several strate-
gies for prevention of origin relicensing, including expression of a
negative regulator known as geminin2,3. Geminin inhibits origin
licensing in S, G2 and M phases through interaction with Cdt1,
thereby blocking MCM loading onto chromatin2–6and thus maintain-
ing genomic stability7–9. Moreover, by physically interacting with tran-
scription factors of the Hox and polycomb families, geminin plays a
major role during early eye and neuronal tube development3,6,10,11. To
understand how the geminin activities are combined in one molecule,
we investigated full-length functional human geminin by transmission
EM of negatively stained protein samples and single-particle analysis.
We first characterized recombinant geminin biochemically and
functionally in a cell-free DNA replication assay12(Fig. 1a,b and
Supplementary Data and Supplementary Methods online). Recom-
binant geminin was fully functional in inhibiting DNA replication ini-
tiation in vitro (Fig. 1a) by blocking Mcm2 loading onto chromatin
(SupplementaryFig. 1 online). The chemical protein crosslinking data
indicated that the main structural organization of geminin is a dimer
that forms a tetramer or dimer of dimers (Fig. 1b). For structural
analysis, we used pH conditions permissive for protein activity. EM
images of geminin show a homogeneous dispersion of small, elon-
gated particles with a bulk at one end (Fig. 1c). A total of 3,300 molec-
ular images were selected manually and subjected to statistical
analysis. Orientations of characteristic views were determined by
angular reconstitution13. The structure of geminin was refined by iter-
ative procedures of alignment and classification, and the final three-
dimensional map of the protein was obtained at a resolution of 17.5 Å
(Supplementary Fig. 2 online). The overall shape of geminin resem-
bles a key (Fig. 2a). The molecule has four visually defined parts: tail,
central body domain, two neck domains and a head domain. The tip
of the tail was not resolved in the reconstruction and may be flexible.
The two necks enclose a large opening with a width of 26.5 Å and a
1Bloomsbury Centre of Structural Biology and 2School of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, UK. 3Wolfson Institute for Biomedical
Research, University College London, Gower Street, London, WC1E 6BT, UK.4Department of Histopathology, Royal Free and University College Medical School,
University College London, London, WC1E 6JJ, UK.5These authors contributed equally to this work. Correspondence should be addressed to G.H.W.
Published online 19 September 2004; doi:10.1038/nsmb835
NATURE STRUCTURAL & MOLECULAR BIOLOGY
VOLUME 11 NUMBER 10 OCTOBER 2004
Figure 1 Characterization and image analysis of geminin. (a) Recombinant
geminin inhibits DNA replication initiation in vitro. NIH 3T3 G1 nuclei were
incubated in elongation buffer (BA) or in extracts from S-phase HeLa cells
(S Cyt), which induce initiation in competent nuclei. Addition of geminin
(Gem) to coincubations of G1 nuclei and S-phase cytosol blocks initiation.
(b) Geminin is an oligomeric protein. Full-length recombinant geminin
was crosslinked with BS3or EGS and analyzed by SDS-PAGE. BS3and
EGS concentrations were increased as indicated by triangles. Units, kDa.
(c) Three-dimensional reconstruction procedure. Orientation angles
β and γ are shown below. (i) Characteristic views of geminin particles
obtained by multireference alignment and classification. (ii) Reprojections
of the geminin structure in the directions found for the views in (i).
(iii) Surface representation of the reconstruction shown in the directions
corresponding to images in (i). Bar, 175 Å. The EM map of geminin
protein and the atomic coordinates of the model have been deposited in
the macromolecular structure database (EBI) under accession codes
EMD-2950 and EBI-20808, respectively.
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol
length of 31 Å. The overall volume of the molecule can accommodate
∼103 kDa of protein mass, corresponding well to four geminin
monomers with a molecular mass of 23.5 kDa each.
Analysis of a truncated form of geminin with deletion of the first 80
residues revealed that the head of the structure represents the N termi-
nus of geminin (Supplementary Fig. 3 online). The combined volume
of the neck and head domains can accommodate ∼64 kDa of poly-
peptide. These data are consistent with the 112-residue N-terminal
part of geminin multiplied by a factor of four, providing evidence for a
tetrameric organization of geminin monomers. Previous studies in
X. laevis mapped the neuronal specification and transcriptional regu-
lation functions of geminin to residues 38–90 (28–79 in human gemi-
nin)2,3. Notably, the hole formed by the head and neck region (Fig. 2)
is large enough to accommodate double-stranded DNA (dsDNA) in a
‘thread in a needle’ style.
A peptide representing residues 112–147 has been crystallized as
a dimeric parallel two-stranded coiled coil14. Because the data are
currently unavailable, we modeled this coiled coil region as a
leucine/isoleucine zipper coordinated into a parallel dimer of
α-helices (Supplementary Fig. 3 online), and fitted this model into
the three-dimensional map of geminin (Fig. 2b). Two dimeric coiled
coil domains fit well into the body domain of geminin, forming a
bouquet-shaped bundle with the N termini facing upward. At the
bottom, the helices appear to form a four-strand coiled coil15
(Fig. 2b), whereas the central portion resembles two conjoined para-
llel three-strand coiled coils15. At the top of the central body, all four
helices lie almost flat in one plane, thus contributing to the observed
flattened shape of this region of geminin. These observations allow us
to assign the central body part of the three-dimensional map to the
coiled coil domain of geminin, with a tetrameric (dimer of dimers)
formation of α-helices.
This structure of geminin, one of the smallest asymmetrical proteins
whose three-dimensional structure has been determined by exploiting
single-particle EM, is coherent with existing biochemical and physio-
logical data, and suggests potential mechanisms of action. There are
two potential binding interfaces for the geminin-Cdt1 interaction.
One is a narrow side of the central body domain with multiple nega-
tive charges on its surface (Supplementary Fig. 3 online), which may
mimic DNA and thus could occupy the DNA-binding pocket of Cdt1
(ref. 16) or other geminin-interacting proteins10,11. The second is the
neck-like segment of the geminin molecule, which in our structure
probably encompasses residues 80–110, previously reported to be crit-
ical for inhibition of replication reinitiation2,3(Fig. 2c). The neck and
body domains could interact with Cdt1 synergistically, with electro-
static contacts being supported by interactions provided by the neck
part of the molecule. Detailed knowledge of geminin’s molecular blue-
print provides new insights into the molecular mechanisms by which
this bifunctional molecule controls cellular proliferation, differentia-
tion and genomic stability.
Note: Supplementary information is available on the Nature Structural & Molecular
We thank A. Dutta for geminin cDNA and H. Saibil, D. Madge and D. Selwood for
stimulating discussions and critical reading of the manuscript. This work has been
funded by Cancer Research UK scientific programme grant SP2360/0103 (G.H.W.
and K.S.) and by UK Biotechnology and Biological Sciences Research Council
programme grant 31/SB09826 (A.L.O., E.V.O., C.B. and U.G.). S.R.K. is supported
by a UK Medical Research Council studentship.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 22 July; accepted 30 August 2004
Published online at http://www.nature.com/nsmb/
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VOLUME 11 NUMBER 10 OCTOBER 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY
Figure 2 Structural organization of the human geminin tetramer. (a) The
key-shaped geminin molecule. From left to right: main, top and side views.
Dimensions are indicated. (b) Three-dimensional map with fitted atomic
models of two coiled coil dimers shown as α-helices. The horizontal cross-
sections depict the internal organization within the four-helix bundle
domain. Right, position of the dimer within the molecular envelope.
(c) Schematic representation of protein density divided into domains with
the protein sequence bar colored accordingly.
© 2004 Nature Publishing Group http://www.nature.com/natstructmolbiol