Structural insights into an atypical histone binding mechanism by a PHD finger.

Complex associating with SET1 (COMPASS) is a histone H3K4 tri-methyltransferase controlled by several regulatory subunits including CXXC zinc finger protein 1 (Cfp1). Prior studies established the structural underpinnings controlling H3K4me3 recognition by the PHD domain of Cfp1's yeast homolog (Spp1). However, metazoans Cfp1PHD lacks structural elements important for H3K4me3 stabilization in Spp1, suggesting that in metazoans, Cfp1PHD domain binds H3K4me3 differently. The structure of Cfp1PHD in complex with H3K4me3 shows unique features such as non-canonical coordination of the first zinc atom and a disulfide bond forcing the reorientation of Cfp1PHD N-terminus, thereby leading to an atypical H3K4me3 binding pocket. This configuration minimizes Cfp1PHD reliance on canonical residues important for histone binding functions of other PHD domains. Cancer-related mutations in Cfp1PHD impair H3K4me3 binding, implying a potential impact on epigenetic signaling. Our work highlights a potential diversification of PHD histone binding modes and the impact of cancer mutations on Cfp1 functions.

Structural analysis of Atopobium parvulum SufS cysteine desulfurase linked to Crohn's disease.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2 S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2 S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.

Analyzing the impact of CFP1 mutational landscape on epigenetic signaling.

CXXC Zinc finger protein 1 (CFP1) is a multitasking protein playing essential roles during various developmental processes. Its ability to interact with several proteins contribute to several epigenetic events. Here, we review CFP1's functions and its impact on DNA methylation and the post-translational modification of histone proteins such as lysine acetylation and methylation. We will also discuss the potential role of CFP1 in carcinogenesis and the impact of the mutations identified in patients suffering from various cancers.

Widespread protein lysine acetylation in gut microbiome and its alterations in patients with Crohn's disease.

Lysine acetylation (Kac), an abundant post-translational modification (PTM) in prokaryotes, regulates various microbial metabolic pathways. However, no studies have examined protein Kac at the microbiome level, and it remains unknown whether Kac level is altered in patient microbiomes. Herein, we use a peptide immuno-affinity enrichment strategy coupled with mass spectrometry to characterize protein Kac in the microbiome, which successfully identifies 35,200 Kac peptides from microbial or human proteins in gut microbiome samples. We demonstrate that Kac is widely distributed in gut microbial metabolic pathways, including anaerobic fermentation to generate short-chain fatty acids. Applying to the analyses of microbiomes of patients with Crohn's disease identifies 52 host and 136 microbial protein Kac sites that are differentially abundant in disease versus controls. This microbiome-wide acetylomic approach aids in advancing functional microbiome research.

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding.

Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5'-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.

Nanoparticle Concentration vs Surface Area in the Interaction of Thiol-Containing Molecules: Toward a Rational Nanoarchitectural Design of Hybrid Materials.

The effect of accounting for the total surface in the association of thiol-containing molecules to nanosilver was assessed using isothermal titration calorimetry, along with a new open access algorithm that calculates the total surface area for samples of different polydispersity. Further, we used advanced molecular dynamic calculations to explore the underlying mechanisms for the interaction of the studied molecules in the presence of a nanosilver surface in the form of flat surfaces or as three-dimensional pseudospherical nanostructures. Our data indicate that not only is the total surface area available for binding but also the supramolecular arrangements of the molecules in the near proximity of the nanosilver surface strongly affects the affinity of thiol-containing molecules to nanosilver surfaces.

A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy.

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.

A charge-suppressing strategy for probing protein methylation.

Methylation of arginine and lysine (RK) residues play essential roles in epigenetics and the regulation of gene expression. However, research in this area is often hindered by the lack of effective tools for probing the protein methylation. Here, we present an antibody-free strategy to capture protein methylation on RK residues by using chemical reactions to eliminate the charges on un-modified RK residues and peptide N-termini. Peptides containing methylated RK residues remain positively charged and are then enriched by strong cation exchange chromatography, followed by high-resolution mass spectrometry identification.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

MFG-E8 Is Critical for Embryonic Stem Cell-Mediated T Cell Immunomodulation.

The molecules and mechanisms pertinent to the low immunogenicity of undifferentiated embryonic stem cells (ESCs) remain poorly understood. Here, we provide evidence that milk fat globule epidermal growth factor 8 (MFG-E8) is a vital mediator in this phenomenon and directly suppresses T cell immune responses. MFG-E8 is enriched in undifferentiated ESCs but diminished in differentiated ESCs. Upregulation of MFG-E8 in ESCs increases the successful engraftment of both undifferentiated and differentiated ESCs across major histocompatibility complex barriers. MFG-E8 suppresses T cell activation/proliferation and inhibits Th1, Th2, and Th17 subpopulations while increasing regulatory T cell subsets. Neutralizing MFG-E8 substantially abrogates these effects, whereas addition of recombinant MFG-E8 to differentiated ESCs restores immunosuppression. Furthermore, we provide the evidence that MFG-E8 suppresses T cell activation and regulates T cell polarization by inhibiting PKCθ phosphorylation through the α3/5ßV integrin receptor. Our findings offer an approach to facilitate transplantation acceptance.

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation.

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.

Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses.

APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G's capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5.

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancer-of-zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers.

The many facets of MLL1 regulation.

In the last 20 years, we have witnessed an exponential number of evidences linking the human mixed lineage leukemia-1 (MLL1) gene to several acute and myelogenous leukemias. MLL1 is one of the founding members of the SET1 family of lysine methyltransferases and is key for the proper control of developmentally regulated gene expression. MLL1 is a structurally complex protein composed of several functional domains. These domains play pivotal roles for the recruitment of regulatory proteins. These MLL1 regulatory proteins (MRPs) dynamically interact with MLL1 and consequently control gene expression. In this review, we summarize recent structural and functional studies of MRPs and discuss emergent structural paradigms for the control of MLL1 activity.

Joint line fullness and meniscal pathology.

BACKGROUND: Meniscal tears have been associated with meniscal cysts and fullness of the knee joint line on physical examination. HYPOTHESIS: Joint line fullness is an accurate, sensitive, and specific test to detect meniscal tears. STUDY DESIGN: Prospective cohort study. METHODS: One hundred consecutive patients undergoing knee arthroscopy were included. All had physical examinations documenting the presence of joint line fullness, joint line tenderness, and the McMurray sign. Arthroscopy was the gold standard for tears. Accuracy, sensitivity, and specificity were calculated and correlated with type of tear. Sixty-one patients had a magnetic resonance imaging preoperatively (the gold standard for determining the presence of a cyst). RESULTS: Meniscal tears were found in 67 patients at arthroscopy. The accuracy, sensitivity, and specificity of joint line fullness were, respectively, 73%, 70%, and 82% in detecting meniscal tears; 68%, 87%, and 30% for joint line tenderness; and 47%, 32%, and 78% for the McMurray sign. The highest positive predictive value for detecting a tear was 88% for joint line fullness, compared with 77% for joint line tenderness and 76% for the McMurray sign. However, joint line fullness did not correlate well with the presence of a cyst, with a low positive predictive value (29%). Of those patients with joint line fullness on physical examination, 89% had a horizontal cleavage component of their tear at arthroscopy. CONCLUSION: Joint line fullness is an accurate, sensitive, and specific test to detect meniscal tears. CLINICAL RELEVANCE: The findings support the routine use of joint line fullness during physical examination along with other common tests to improve the accuracy of clinically diagnosing meniscal tears.

The plasticity of WDR5 peptide-binding cleft enables the binding of the SET1 family of histone methyltransferases.

In mammals, the SET1 family of lysine methyltransferases (KMTs), which includes MLL1-5, SET1A and SET1B, catalyzes the methylation of lysine-4 (Lys-4) on histone H3. Recent reports have demonstrated that a three-subunit complex composed of WD-repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5) and absent, small, homeotic disks-2-like (ASH2L) stimulates the methyltransferase activity of MLL1. On the basis of studies showing that this stimulation is in part controlled by an interaction between WDR5 and a small region located in close proximity of the MLL1 catalytic domain [referred to as the WDR5-interacting motif (Win)], it has been suggested that WDR5 might play an analogous role in scaffolding the other SET1 complexes. We herein provide biochemical and structural evidence showing that WDR5 binds the Win motifs of MLL2-4, SET1A and SET1B. Comparative analysis of WDR5-Win complexes reveals that binding of the Win motifs is achieved by the plasticity of WDR5 peptidyl-arginine-binding cleft allowing the C-terminal ends of the Win motifs to be maintained in structurally divergent conformations. Consistently, enzymatic assays reveal that WDR5 plays an important role in the optimal stimulation of MLL2-4, SET1A and SET1B methyltransferase activity by the RbBP5-ASH2L heterodimer. Overall, our findings illustrate the function of WDR5 in scaffolding the SET1 family of KMTs and further emphasize on the important role of WDR5 in regulating global histone H3 Lys-4 methylation.

Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2.

The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction network for three different human SMYD proteins (SMYD2, SMYD3, and SMYD5). Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins. Among those, we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3. Moreover, using proteomic and biochemical techniques, we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains, respectively. In addition, we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones, pH, and demethylation by the lysine amine oxidase LSD1, suggesting that alternative mechanisms control HSP90 methylation by SMYD2. Altogether, this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.

Structural and biochemical insights into MLL1 core complex assembly.

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5's ß-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.

Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic.

The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an Nα acetylated form of histone H3 peptide (NαH3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of NαH3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.

SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation.

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.