An "Onion-like" Model of Protein Unfolding: Collective versus Site Specific Approaches.

Approximating protein unfolding by an all-or-none cooperative event is a convenient assumption that can provide precious global information on protein stability. It is however quickly emerging that the scenario is far more complex and that global denaturation curves often hide a rich heterogeneity of states that are largely probe dependent. In this review, we revisit the importance of gaining site-specific information on the unfolding process. We focus on nuclear magnetic resonance, as this is the main technique able to provide site-specific information. We review historical and most modern approaches that have allowed an appreciable advancement of the field of protein folding. We also demonstrate how unfolding is a reporter dependent event, suggesting the outmost importance of selecting the reporter carefully.

Distinct roles of haspin in stem cell division and male gametogenesis.

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide.

Protein phosphorylation is a key regulatory mechanism in eukaryotic cells. In the intrinsically disordered histone tails, phosphorylation is often a part of combinatorial post-translational modifications and an integral part of the "histone code" that regulates gene expression. Here, we study the association between two histone H3 tail peptides modified to different degrees, using fully atomistic molecular dynamics simulations. Assuming that the initial conformations are either α-helical or fully extended, we compare the propensity of the two peptides to associate with one another when both are unmodified, one modified and the other unmodified, or both modified. The simulations lead to the identification of distinct inter- and intramolecular interactions in the peptide dimer, highlighting a prominent role of a fine-tuned phosphorylation rheostat in peptide association. Progressive phosphorylation appears to modulate peptide charge, inducing strong and specific intermolecular interactions between the monomers, which do not result in the formation of amorphous or ordered aggregates, as documented by experimental evidence derived from Circular Dichroism and NMR spectroscopy. However, upon complete saturation of positive charges by phosphate groups, this effect is reversed: intramolecular interactions prevail and dimerization of zero-charge peptides is markedly reduced. These findings underscore the role of phosphorylation thresholds in the dynamics of intrinsically disordered proteins. Phosphorylation rheostats might account for the divergent effects of histone modifications on the modulation of chromatin structure.

Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation.

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

Heterochromatin remodeling in embryonic stem cells proceeds through stochastic de-stabilization of regional steady-states.

Cell differentiation is associated with progressive immobilization of chromatin proteins, expansion of heterochromatin, decrease of global transcriptional activity and induction of lineage-specific genes. However, how these processes relate to one another remains unknown. We show here that the heterochromatic domains of mouse embryonic stem cells (ESCs) are dynamically distinct and possesses a mosaic sub-structure. Although random spatio-temporal fluctuations reshuffle continuously the chromatin landscape, each heterochromatic territory maintains its dynamic profile, exhibiting robustness and resembling a quasi-steady state. Transitions towards less dynamic states are detected sporadically as ESCs downregulate Nanog and exit the self-renewal phase. These transitions increase in frequency after lineage-commitment, but evolve differently depending on cellular context and transcriptional status. We propose that chromatin remodeling is a step-wise process, which involves stochastic de-stabilization of regional steady states and formation of new dynamic ensembles in coordination to changes in the gene expression program.

The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

Structural role of RKS motifs in chromatin interactions: a molecular dynamics study of HP1 bound to a variably modified histone tail.

The current understanding of epigenetic signaling assigns a central role to post-translational modifications that occur in the histone tails. In this context, it has been proposed that methylation of K9 and phosphorylation of S10 in the tail of histone H3 represent a binary switch that controls its reversible association to heterochromatin protein 1 (HP1). To test this hypothesis, we performed a comprehensive molecular dynamics study in which we analyzed a crystallographically defined complex that involves the HP1 chromodomain and an H3 tail peptide. Microsecond-long simulations show that the binding of the trimethylated K9 H3 peptide in the aromatic cage of HP1 is only slightly affected by S10 phosphorylation, because the modified K9 and S10 do not interact directly with one another. Instead, the phosphate group of S10 seems to form a persistent intramolecular salt bridge with R8, an interaction that can provoke a major structural change and alter the hydrogen-bonding regime in the H3-HP1 complex. These observations suggest that interactions between adjacent methyl-lysine and phosphoserine side chains do not by themselves provide a binary switch in the H3-HP1 system, but arginine-phosphoserine interactions, which occur in both histones and nonhistone proteins in the context of a conserved RKS motif, are likely to serve a key regulatory function.

Solution structure and molecular interactions of lamin B receptor Tudor domain.

Lamin B receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear membrane with the underlying nuclear lamina and peripheral heterochromatin. To better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harbor a Tudor domain (LBR-TD). Structural analysis by multidimensional NMR spectroscopy establishes that LBR-TD indeed adopts a classical ß-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more mobile at the plane of the nuclear envelope, but the isolated module does not bind to nuclear lamins, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhibits tight and stoichiometric binding to the "histone-fold" region of unassembled, free histone H3, suggesting an interesting role in histone assembly. Consistent with such a role, robust binding to native nucleosomes is observed when LBR-TD is extended toward its carboxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in combination with LBR-TD, binds both unassembled and assembled H3/H4 histones, suggesting that the TD/RS interface may operate as a "histone chaperone-like platform."

ERBIN is a new SARA-interacting protein: competition between SARA and SMAD2 and SMAD3 for binding to ERBIN.

SARA, an early endosomal protein, plays a key role in TGFß signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFß receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208-1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA- and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFß and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.

Casein kinase 1 regulates human hypoxia-inducible factor HIF-1.

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits.

Phosphorylation of histone H3 at Thr3 is part of a combinatorial pattern that marks and configures mitotic chromatin.

We have previously shown that histone H3 is transiently phosphorylated at Thr3 during mitosis. Extending these studies, we now report that phosphorylated Thr3 is always in cis to trimethylated Lys4 and dimethylated Arg8, forming a new type of combinatorial modification, which we have termed PMM. PMM-marked chromatin emerges at multiple, peripheral sites of the prophase nucleus, then forms distinct clusters at the centric regions of metaphase chromosomes, and finally spreads (as it wanes) to the distal areas of segregating chromatids. The characteristic prophase pattern can be reproduced by expressing ectopically the kinase haspin at interphase, suggesting that the formation of the PMM signature does not require a pre-existing mitotic environment. On the other hand, the ;dissolution' and displacement of PMM clusters from a centric to distal position can be induced by partial dephosphorylation or chromosome unravelling, indicating that these changes reflect the regulated grouping and scrambling of PMM subdomains during cell division. Formation of PMM is prevented by haspin knockdown and leads to delayed exit from mitosis. However, PMM-negative cells do not exhibit major chromosomal defects, suggesting that the local structures formed by PMM chromatin may serve as a ;licensing system' that allows quick clearance through the metaphase-anaphase checkpoint.

Identification of distinct SET/TAF-Ibeta domains required for core histone binding and quantitative characterisation of the interaction.

BACKGROUND: The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Ibeta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Ibeta, we designed several SET/TAF-Ibeta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. RESULTS: Wild type SET/TAF-Ibeta binds to histones H2B and H3 with Kd values of 2.87 and 0.15 microM, respectively. The preferential binding of SET/TAF-Ibeta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Ibeta, as well as the H3 amino-terminal tail, are dispensable for this interaction. CONCLUSION: This type of analysis allowed us to assess the relative affinities of SET/TAF-Ibeta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Ibeta and can be valuable to understand the role of SET/TAF-Ibeta in chromatin function.

Chromatin remodeling during mitosis: a structure-based code?

Histone modifications have been associated with particular states of transcriptional activity and are thought to serve as an "information code". However, this principle does not apply to histone phosphorylation, which can be detected in two, seemingly contrasting situations, i.e., in a transcriptionally hyperactive state following growth factor stimulation and in transcriptionally paused mitotic chromosomes. There are several indications that mitotic phosphorylation of histone H3 at serine-10 by the Aurora B kinase and trimethylation at lysine-9 by the methyl transferase Suvar3,9 operate as a "binary switch", which determines recruitment or eviction of heterochromatin-specific proteins from pericentromeric repeats. Moreover, threonine-3 phosphorylation of histone H3 by the newly identified haspin kinase seems to promote chromatid cohesion during mitosis. We discuss here emerging information and new ideas suggesting that these modifications, in combination to upstream and downstream marks, constitute a system of intrinsic folding determinants that facilitate chromatin condensation and confer topological specificity to mitotic chromosomes.

An automated method for gridding in microarray images.

Microarray technology is a powerful tool for analyzing the expression of a large number of genes in parallel. A typical microarray image consists of a few thousands of spots which determine the level of gene expression in the sample. In this paper we propose a method which automatically addresses each spot area in the image. Initially, a preliminary segmentation of the image is produced using a template matching algorithm. Next, grid and spot finding are realized. The position of non-expressed spots is located and finally a Voronoi diagram is employed to fit the grid on the image. Our method has been evaluated in a set of five images consisting of 45960 spots, from the Stanford microarray database and the reported accuracy for spot detection was 93%

Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure.

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.

Human recombinant mutated forms of the mitochondrial COX assembly Sco2 protein differ from wild-type in physical state and copper binding capacity.

The human Sco2 protein is a cytochrome c oxidase assembly protein that participates in mitochondrial copper pathway, acting downstream of Cox17 protein. In a previous work, we detected mutations in the human SCO2 gene in three unrelated infants with fatal cardioencephalomyopathy and COX deficiency. In this study, full-length processed recombinant wild-type and two mutated forms of hSco2p (w/t-rhSco2p, E140K-rhSco2p, and S225F-rhSco2p) were produced in bacteria as soluble recombinant peptides for the first time and evaluated for differences in their physical state and ability to bind copper. Our data indicate the following: (a) w/t-rhSco2p and S225F-rhSco2p were found to be in a monomeric form in contrast to E140K-rhSco2p that was in a major non-reducible dimer and a minor monomer form; (b) wild-type and mutated rhSco2p exhibited clear differences in their physical conformational state, as shown by circular dichroism and thermal denaturation analyses; (c) copper binding studies showed that E140K-rhSco2p bound markedly less copper while S225F-rhSco2p more than expected as compared to amount of the copper bound with w/t-rhSco2p. rhCox17p served as positive control experiment. These data indicate that S225F and E140K mutations found in the SCO2 gene derived from patients alter the physical conformational state of encoded hSco2p that may disturb the normal copper transport pathway in mitochondria. These findings are valuable for understanding the molecular basis of fatal cardioencephalomyopathy and COX deficiency and for designing appropriate pharmacological interventions.

Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement.

Confinement of a protein in a small inert space and microviscosity are known to increase its thermodynamic stability in a way similar to the mechanisms that stabilize protein fold in the cell. Here, to examine the influence of confinement on protein stability we choose four test cases of single domain proteins characterized by a wide range of melting temperatures, from approximately 73 degrees C of titin I27 to approximately 36 degrees C of yeast frataxin. All proteins are stabilized when confined in the gel, the most dramatic stabilization being that of yeast frataxin, whose melting temperature increased by almost 5 degrees C in the gel. In addition to being simple to use, this approach allows us to change the viscosity of the solvent without changing its composition or altering the structure of the proteins. The dimensions of the pores of the gels fall in the nanometer range, hence they are similar to those of the chaperone cavity. This method could therefore be used as a novel and powerful approach for protein folding studies.

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization.

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.

The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO.

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.

Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain.

SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain. SecA recognizes mature and signal peptide preprotein regions. We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding. Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain. SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins. Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch. IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity. This mechanism coordinates signal peptide binding with ATPase activation. Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase. This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants. Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.