VprBP/DCAF1 Triggers Melanomagenic Gene Silencing through Histone H2A Phosphorylation.

Vpr binding protein (VprBP), also known as DDB1- and CUL4-associated factor1 (DCAF1), is a recently identified atypical kinase and plays an important role in downregulating the transcription of tumor suppressor genes as well as increasing the risk for colon and prostate cancers. Melanoma is the most aggressive form of skin cancer arising from pigment-producing melanocytes and is often associated with the dysregulation of epigenetic factors targeting histones. Here, we demonstrate that VprBP is highly expressed and phosphorylates threonine 120 (T120) on histone H2A to drive the transcriptional inactivation of growth-regulatory genes in melanoma cells. As is the case for its epigenetic function in other types of cancers, VprBP acts to induce a gene silencing program dependent on H2AT120 phosphorylation (H2AT120p). The significance of VprBP-mediated H2AT120p is further underscored by the fact that VprBP knockdown- or VprBP inhibitor-induced lockage of H2AT120p mitigates melanoma tumor growth in xenograft models. Collectively, our results establish VprBP-mediated H2AT120p as a key epigenetic signal for melanomagenesis and suggest the therapeutic potential of targeting VprBP kinase activity for effective melanoma treatment.

Comparison of Integrin αIIbß3 Transmembrane Association in Vesicles and Bicelles.

Membrane proteins are commonly reconstituted in membrane mimics exhibiting discontinuous lipid bilayers. In contrast, the continuous membranes of cells are conceptually best represented by large unilamellar vesicles (LUVs). Here, we compared the thermodynamic stability of the integrin αIIbß3 transmembrane (TM) complex between vesicles and bicelles to assess the consequence of this simplification. In LUVs, we further evaluated the strength of the αIIb(G972S)-ß3(V700T) interaction that corresponds to the hydrogen bond interaction postulated for ß2 integrins. An upper limit of 0.9 kcal/mol was estimated for superior TM complex stabilization in LUVs relative to bicelles. Compared to the αIIbß3 TM complex stability in LUVs of 5.6 ± 0.2 kcal/mol, this limit is modest, indicating that bicelles performed well relative to LUVs. The implementation of ß3(V700T) alleviated αIIb(G972S) destabilization by 0.4 ± 0.2 kcal/mol in confirmation of relatively weak hydrogen bonding. Interestingly, the hydrogen bond adjusts the TM complex stability to a level that is not achievable by merely varying the residue corresponding to αIIb(Gly972).

VprBP/DCAF1 triggers melanomagenic gene silencing through histone H2A phosphorylation.

Background: Melanoma is the most aggressive form of skin cancer arising from pigment-producing melanocytes and is often associated with dysregulation of epigenetic factors targeting histones. VprBP, also known as DCAF1, is a recently identified kinase and plays an important role in downregulating the transcription of tumor suppressor genes as well as increasing the risk for colon and prostate cancers. However, it remains unknown whether VprBP is also involved in triggering the pathogenesis of other types of cancer. Results: We demonstrate that VprBP is highly expressed and phosphorylates threonine 120 (T120) on histone H2A to drive transcriptional inactivation of growth regulatory genes in melanoma cells. As is the case for its epigenetic function in colon and prostate cancers, VprBP acts to induce gene silencing program dependently of H2AT120 phosphorylation (H2AT120p). The significance of VprBP-mediated H2AT120p is further underscored by the fact that VprBP knockdown- or VprBP inhibitor-induced lockage of H2AT120p mitigates melanoma tumor growth in xenograft models. Moreover, artificial tethering of VprBP wild type, but not VprBP kinase-dead mutant, to its responsive genes is sufficient for achieving an inactive transcriptional state in VprBP-depleted cells, indicating that VprBP drives gene silencing program in an H2AT120p-dependent manner. Conclusions: Our results establish VprBP-mediated H2AT120p as a key epigenetic signal for melanomagenesis and suggest the therapeutic potential of targeting VprBP kinase activity for effective melanoma treatment.

Phosphorylation and stabilization of EZH2 by DCAF1/VprBP trigger aberrant gene silencing in colon cancer.

Our recent work has shown that DCAF1 (also known as VprBP) is overexpressed in colon cancer and phosphorylates histone H2AT120 to drive epigenetic gene inactivation and oncogenic transformation. We have extended these observations by investigating whether DCAF1 also phosphorylates non-histone proteins as an additional mechanism linking its kinase activity to colon cancer development. We now demonstrate that DCAF1 phosphorylates EZH2 at T367 to augment its nuclear stabilization and enzymatic activity in colon cancer cells. Consistent with this mechanistic role, DCAF1-mediated EZH2 phosphorylation leads to elevated levels of H3K27me3 and altered expression of growth regulatory genes in cancer cells. Furthermore, our preclinical studies using organoid and xenograft models revealed that EZH2 requires phosphorylation for its oncogenic function, which may have therapeutic implications for gene reactivation in colon cancer cells. Together, our data define a mechanism underlying DCAF1-driven colonic tumorigenesis by linking DCAF1-mediated EZH2 phosphorylation to EZH2 stability that is crucial for establishing H3K27me3 and gene silencing program.

Molecular basis of Q-length selectivity for the MW1 antibody-huntingtin interaction.

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

Isothermal Titration Calorimetry of Membrane Proteins.

The ability to quantify protein-protein interactions without adding labels to protein has made isothermal titration calorimetry (ITC) a preferred technique to study proteins in aqueous solution. Here, we describe the application of ITC to the study of protein-protein interactions in membrane mimics using the association of integrin αIIb and ß3 transmembrane domains in phospholipid bicelles as an example. A higher conceptual and experimental effort compared to water-soluble proteins is required for membrane proteins and rewarded with rare thermodynamic insight into this central class of proteins.

Insight Into Pathological Integrin αIIbß3 Activation From Safeguarding The Inactive State.

The inhibition of physiological activation pathways of the platelet adhesion receptor integrin αIIbß3 may fail to prevent fatal thrombosis, suggesting that the receptor is at risk of activation by yet an unidentified pathway. Here, we report the discovery and characterization of a structural motif that safeguards the receptor by selectively destabilizing its inactive state. At the extracellular membrane border, an overpacked αIIb(W968)-ß3(I693) contact prevents αIIb(Gly972) from optimally assembling the αIIbß3 transmembrane complex, which maintains the inactive state. This destabilization of approximately 1.0 kcal/mol could be mitigated by hydrodynamic forces but not physiological agonists, thereby identifying hydrodynamic forces as pathological activation stimulus. As reproductive life spans are not generally limited by cardiovascular disease, it appears that the evolution of the safeguard was driven by fatal, hydrodynamic force-mediated integrin αIIbß3 activation in the healthy cardiovascular system. The triggering of the safeguard solely by pathological stimuli achieves an effective increase of the free energy barrier between inactive and active receptor states without incurring an increased risk of bleeding. Thus, integrin αIIbß3 has evolved an effective way to protect receptor functional states that indicates the availability of a mechanical activation pathway when hydrodynamic forces exceed physiological margins.

Topological Adaptation of Transmembrane Domains to the Force-Modulated Lipid Bilayer Is a Basis of Sensing Mechanical Force.

Cells can sense and respond to various mechanical stimuli from their surrounding environment. One of the explanations for mechanosensitivity, a lipid-bilayer model, suggests that a stretch of the membrane induced by mechanical force alters the physical state of the lipid bilayer, driving mechanosensors to assume conformations better matched to the altered membrane. However, mechanosensors of this class are restricted to ion channels. Here, we reveal that integrin αIIbß3, a prototypic adhesion receptor, can be activated by various mechanical stimuli including stretch, shear stress, and osmotic pressure. The force-induced integrin activation was not dependent on its known intracellular activation signaling events and was even observed in reconstituted cell-free liposomes. Instead, these mechanical stimuli were found to alter the lipid embedding of the integrin ß3 transmembrane domain (TMD) and subsequently weaken the αIIb-ß3 TMD interaction, which results in activation of the receptor. Moreover, artificial modulation of the membrane curvature near integrin αIIbß3 can induce its activation in cells as well as in lipid nanodiscs, suggesting that physical deformation of the lipid bilayer, either by mechanical force or curvature, can induce integrin activation. Thus, our results establish the adhesion receptor as a bona fide mechanosensor that directly senses and responds to the force-modulated lipid environment. Furthermore, this study expands the lipid-bilayer model by suggesting that the force-induced topological change of TMDs and subsequent alteration in the TMD interactome is a molecular basis of sensing mechanical force transmitted via the lipid bilayer.

Universal principles of membrane protein assembly, composition and evolution.

Structural diversity in α-helical membrane proteins (MP) arises from variations in helix-helix crossings and contacts that may bias amino acid usage. Here, we reveal systematic changes in transmembrane amino acid frequencies (f) as a function of the number of helices (n). For eukarya, breaks in f(n) trends of packing (Ala, Gly and Pro), polar, and hydrophobic residues identify different MP assembly principles for 2≤n≤7, 8≤n≤12 and n≥13. In bacteria, the first f break already occurs after n = 6 in correlation to an earlier n peak in MP size distribution and dominance of packing over polar interactions. In contrast to the later n brackets, the integration levels of helix bundles continuously increased in the first, most populous brackets indicating the formation of single structural units (domains). The larger first bracket of eukarya relates to a balance of polar and packing interactions that enlarges helix-helix combinatorial possibilities (MP diversity). Between the evolutionary old, packing and new, polar residues f anti-correlations extend over all biological taxa, broadly ordering them according to evolutionary history and allowing f estimates for the earliest forms of life. Next to evolutionary history, the amino acid composition of MP is determined by size (n), proteome diversity, and effective amino acid cost.

p32 is a negative regulator of p53 tetramerization and transactivation.

p53 is a sequence-specific transcription factor, and proper regulation of p53 transcriptional activity is critical for orchestrating different tumor-suppressive mechanisms. p32 is a multifunctional protein which interacts with a large number of viral proteins and transcription factors. Here, we investigate the effect of p32 on p53 transactivation and identify a novel mechanism by which p32 alters the functional characteristics of p53. Specifically, p32 attenuates p53-dependent transcription through impairment of p53 binding to its response elements on target genes. Upon p32 expression, p53 levels bound at target genes are decreased, and p53 target genes are inactivated, strongly indicating that p32 restricts p53 occupancy and function at target genes. The primary mechanism contributing to the observed action of p32 is the ability of p32 to interact with the p53 tetramerization domain and to block p53 tetramerization, which in turn enhances nuclear export and degradation of p53, leading to defective p53 transactivation. Collectively, these data establish p32 as a negative regulator of p53 function and suggest the therapeutic potential of targeting p32 for cancer treatment.

The folding equilibrium of huntingtin exon 1 monomer depends on its polyglutamine tract.

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

Regulation of Breast Cancer-Induced Osteoclastogenesis by MacroH2A1.2 Involving EZH2-Mediated H3K27me3.

Breast cancer cells relocate to bone and activate osteoclast-induced bone resorption. Soluble factors secreted by breast cancer cells trigger a cascade of events that stimulate osteoclast differentiation in the bone microenvironment. MacroH2A is a unique histone variant with a C-terminal non-histone domain and plays a crucial role in modulating chromatin organization and gene transcription. Here, we show that macroH2A1.2, one of the macroH2A isoforms, has an intrinsic ability to inhibit breast cancer-derived osteoclastogenesis. This repressive effect requires macroH2A1.2-dependent attenuation of expression and secretion of lysyl oxidase (LOX) in breast cancer cells. Furthermore, our mechanistic studies reveal that macroH2A1.2 physically and functionally interacts with the histone methyltransferase EZH2 and elevates H3K27me3 levels to keep LOX gene in a repressed state. Collectively, this study unravels a role for macroH2A1.2 in regulating osteoclastogenic potential of breast cancer cells, suggesting possibilities for developing therapeutic tools to treat osteolytic bone destruction.

H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis.

BACKGROUND: MMP-9 plays a direct role in the activation of pro-osteoclastogenic genes by cleaving histone H3N-terminal tail (H3NT) and altering chromatin architecture. Although H3 acetylation at K18 has been shown to stimulate MMP-9 enzymatic activity toward H3NT, nothing is known about the influence of other H3NT modifications on this epigenetic reaction. RESULTS: We show that H3 monomethylation at lysine 27 (H3K27me1) is essential for MMP-9-dependent H3NT proteolysis during RANKL-induced osteoclast differentiation. Through the recognition of H3K27me1 mark, MMP-9 localizes and generates H3NT proteolysis at the genes encoding osteoclast differentiation factors. By using RNAi and small molecule inhibitor approaches, we also confirmed that G9a is the major methyltransferase to catalyze H3K27me1 for MMP-9-dependent H3NT proteolysis and trigger the expression of osteoclast-specific genes. CONCLUSIONS: Our data establish new functions for G9a-mediated H3K27me1 in MMP-9-dependent H3NT proteolysis and demonstrate how histone modification can be exploited to regulate osteoclastogenic gene expression at the molecular level. Further studies are warranted to investigate the detailed mechanism by which G9a overexpression with concomitant dysregulation of osteoclastogenesis contributes to the pathogenesis of bone disorders.

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbß3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.

Direct Evaluation of Protein-Lipid Contacts Reveals Protein Membrane Immersion and Isotropic Bicelle Structure.

The solvation of membrane proteins by both lipids and water makes their membrane immersion difficult to predict and the choice of a membrane mimic challenging. To characterize protein-lipid contacts and bicelle membrane mimics, we examined protein-lipid cross-relaxation of integrin αIIb and ß3(A711P) transmembrane helices in isotropic phospholipid bicelles (q = 0.5 and 0.7). Long-chain bicelle lipids dominated contacts with central helix segments, whereas both short- and long-chain lipids contacted the terminal turns of each helix in corroboration of the mixed bicelle model. The saturation transfer profiles from long-chain lipids directly established helix midpoints in the lipid bilayer. Lipid headgroups and water molecules engaged the side chains of buried serine and threonine in competition with intrahelical hydrogen bonding, illustrating that polar side chains seek the most favorable electrostatic contacts.

Structural and thermodynamic basis of proline-induced transmembrane complex stabilization.

In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbß3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the ß3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of -0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbß3(A711P).

A Conserved Ectodomain-Transmembrane Domain Linker Motif Tunes the Allosteric Regulation of Cell Surface Receptors.

In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit ß and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors.

MMP-9 facilitates selective proteolysis of the histone H3 tail at genes necessary for proficient osteoclastogenesis.

Although limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, the specific genomic sites targeted for H3NT proteolysis and the functional significance of H3NT cleavage remain largely unknown. Here we report the first method to identify and examine H3NT-cleaved regions in mammals, called chromatin immunoprecipitation (ChIP) of acetylated chromatin (ChIPac). By applying ChIPac combined with deep sequencing (ChIPac-seq) to an established cell model of osteoclast differentiation, we discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most H3NT-cleaved genes displayed significant expression changes during osteoclastogenesis. We also discovered that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). In contrast to other known H3NT proteases, MMP-9 primarily cleaved H3K18-Q19 in vitro and in cells. Furthermore, our results support CBP/p300-mediated acetylation of H3K18 as a central regulator of MMP-9 H3NT protease activity both in vitro and at H3NT cleavage sites during osteoclastogenesis. Importantly, we found that abrogation of H3NT proteolysis impaired osteoclastogenic gene activation concomitant with defective osteoclast differentiation. Our collective results support the necessity of MMP-9-dependent H3NT proteolysis in regulating gene pathways required for proficient osteoclastogenesis.

Linker histone H1.2 establishes chromatin compaction and gene silencing through recognition of H3K27me3.

Linker histone H1 is a protein component of chromatin and has been linked to higher-order chromatin compaction and global gene silencing. However, a growing body of evidence suggests that H1 plays a gene-specific role, regulating a relatively small number of genes. Here we show that H1.2, one of the H1 subtypes, is overexpressed in cancer cells and contributes to gene silencing. H1.2 gets recruited to distinct chromatin regions in a manner dependent on EZH2-mediated H3K27me3, and inhibits transcription of multiple growth suppressive genes via modulation of chromatin architecture. The C-terminal tail of H1.2 is critical for the observed effects, because mutations of three H1.2-specific amino acids in this domain abrogate the ability of H1.2 to bind H3K27me3 nucleosomes and inactivate target genes. Collectively, these results provide a molecular explanation for H1.2 functions in the regulation of chromatin folding and indicate that H3K27me3 is a key mechanism governing the recruitment and activity of H1.2 at target loci.