N-terminal Regulatory Domain Research Articles

How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

Neutralization and Stability of JN.1-derived LB.1, KP.2.3, KP.3 and KP.3.1.1 Subvariants.

During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants-LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.

Structural insight into the DNMT1 reaction cycle by cryo-electron microscopy.

DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1's N-terminal regulatory domains during the transition from an apo 'auto-inhibited' to a DNA-bound 'non-productive' and finally a DNA-bound 'productive' state of DNMT1. Furthermore, we address the regulation of DNMT1's methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a 'non-productive' state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1.

The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

LGG-26. A ROLE FOR BRAF FUSION PARTNERS IN ONCOGENIC SIGNALING

Abstract t was originally hypothesized that loss of BRAF N-terminal negative regulatory domains, which are deleted in the KIAA1549:BRAF rearrangement, results in constitutive activation of the BRAF kinase. This model suggests that BRAF fusion partners are dispensable for BRAF oncogenic signaling. Paradoxically, our data suggest expression of truncated BRAF is insufficient for transformation and identify specific domains in KIAA1549 that are necessary for transformation. These domains are critical for regulating subcellular localization of the fusion protein and highlight an aberrant pattern of cellular localization that is not observed with wildtype BRAF. Altered subcellular localization results in proteolytic cleavage of KIAA1549:BRAF, and we identify the protease responsible for this cleavage event. This protease is therapeutically tractable and clinically relevant inhibitors have been developed, presenting a MAPK-independent mechanism to target the fusion. We also highlight an unexpected role for rare fusion partners, including FAM131B, in BRAF activation. In total, these data suggest that BRAF fusion partners are not indispensable for transformation as was previously thought, presenting unexplored opportunities to therapeutically target pLGG tumors with BRAF rearrangements. We are currently working on the IP measures required to disclose the specific enzyme(s) at ISPNO.

Androgen Signaling in Prostate Cancer: When a Friend Turns Foe.

Androgen (AR) signaling is the main signaling for the development of the prostate and its normal functioning. AR is highly specific for testosterone and dihydrotestosterone, significantly contributing to prostate development, physiology, and cancer. All these receptors have emerged as crucial therapeutic targets for PCa. In the year 1966, the Noble prize was awarded to Huggins and Hodge for their groundbreaking discovery of AR. As it is a pioneer transcription factor, it belongs to the steroid hormone receptor family and consists of domains, including DNA binding domain (DBD), hormone response elements (HRE), C-terminal ligand binding domain (LBD), and N-terminal regulatory domains. Structural variations in AR, such as AR gene amplification, LBD mutations, alternative splicing of exons, hypermethylation of AR, and co- regulators, are major contributors to PCa. It's signaling is crucial for the development and functioning of the prostate gland, with the AR being the key player. The specificity of AR for testosterone and dihydrotestosterone is important in prostate physiology. However, when it is dysregulated, AR contributes significantly to PCa. However, the structural variations in AR, such as gene amplification, mutations, alternative splicing, and epigenetic modifications, drive the PCa progression. Therefore, understanding AR function and dysregulation is essential for developing effective therapeutic strategies. Thus, the aim of this review was to examine how AR was initially pivotal for prostate development and how it turned out to show both positive and detrimental implications for the prostate.

Genome-wide characterization of DNA methyltransferase family genes implies GhDMT6 improving tolerance of salt and drought on cotton

BackgroundDNA methylation is an important epigenetic mode of genomic DNA modification and plays a vital role in maintaining epigenetic content and regulating gene expression. Cytosine-5 DNA methyltransferase (C5-MTase) are the key enzymes in the process of DNA methylation. However, there is no systematic analysis of the C5-MTase in cotton so far, and the function of DNMT2 genes has not been studied.MethodsIn this study, the whole genome of cotton C5-MTase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome, and the function of GhDMT6 was further validated by VIGS experiments and subcellular localization analysis.Results33 C5-MTases were identified from three cotton genomes, and were divided into four subfamilies by systematic evolutionary analysis. After the protein domain alignment of C5-MTases in cotton, 6 highly conserved motifs were found in the C-terminus of 33 proteins involved in methylation modification, which indicated that C5-MTases had a basic catalytic methylation function. These proteins were divided into four classes based on the N-terminal difference, of which DNMT2 lacks the N-terminal regulatory domain. The expression of C5-MTases in different parts of cotton was different under different stress treatments, which indicated the functional diversity of cotton C5-MTase gene family. Among the C5-MTases, the GhDMT6 had a obvious up-regulated expression. After silencing GhDMT6 with VIGS, the phenotype of cotton seedlings under different stress treatments showed a significant difference. Compared with cotton seedlings that did not silence GhDMT6, cotton seedlings silencing GhDMT6 showed significant stress resistance.ConclusionThe results show that C5-MTases plays an important role in cotton stress response, which is beneficial to further explore the function of DNMT2 subfamily genes.

Ca2+/CaM dependent protein kinase II (CaMKII)α and CaMKIIβ hub domains adopt distinct oligomeric states and stabilities.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a multidomain serine/threonine kinase that plays important roles in the brain, heart, muscle tissue, and eggs/sperm. The N-terminal kinase and regulatory domain is connected by a flexible linker to the C-terminal hub domain. The hub domain drives the oligomeric organization of CaMKII, assembling the kinase domains into high local concentration. Previous structural studies have shown multiple stoichiometries of the holoenzyme as well as the hub domain alone. Here, we report a comprehensive study of the hub domain stoichiometry and stability in solution. We solved two crystal structures of the CaMKIIβ hub domain that show 14-mer (3.1 Å) and 16-mer (3.4 Å) assemblies. Both crystal structures were determined from crystals grown in the same drop, which suggests that CaMKII oligomers with different stoichiometries likely coexist. To further interrogate hub stability, we employed mass photometry and temperature denaturation studies of CaMKIIβ and CaMKIIα hubs, which highlight major differences between these highly similar domains. We created a dimeric CaMKIIβ hub unit using rational mutagenesis, which is significantly less stable than the oligomer. Both hub domains populate an intermediate during unfolding. We found that multiple CaMKIIβ hub stoichiometries are present in solution and that larger oligomers are more stable. CaMKIIα had a narrower distribution of molecular weight and was distinctly more stable than CaMKIIβ.

Neutralization of SARS-CoV-2 Omicron subvariant BA.2.87.1

A new highly mutated Omicron subvariant BA.2.87.1 has recently been identified with over 30 amino acid mutations in the Spike protein compared with BA.2, BA.5, XBB.1.5, and JN.1 variants. Mutiple mutations in BA.2.87.1 are located in the N-terminal domain (NTD) rather than in the receptor binding domain (RBD) of the Spike protein. We evaluated neutralizing antibody (NAb) responses to BA.2.87.1 because of its highly mutated sequence and its unique NTD region. Our data show that NAb responses to BA.2.87.1 were lower than to BA.2 but higher than to JN.1, suggesting that BA.2.87.1 is not a further antibody escape variant compared with other currently circulating variants. Moreover, XBB.1.5 mRNA boosting increased NAb titers to all variants tested including BA.2.87.1.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

The Complement-Targeted Inhibitor Mini-FH Protects against Experimental Periodontitis via Both C3-Dependent and C3-Independent Mechanisms.

A minimized version of complement factor H (FH), designated mini-FH, was previously engineered combining the N-terminal regulatory domains (short consensus repeat [SCR]1-4) and C-terminal host-surface recognition domains (SCR19-20) of the parent molecule. Mini-FH conferred enhanced protection, as compared with FH, in an ex vivo model of paroxysmal nocturnal hemoglobinuria driven by alternative pathway dysregulation. In the current study, we tested whether and how mini-FH could block another complement-mediated disease, namely periodontitis. In a mouse model of ligature-induced periodontitis (LIP), mini-FH inhibited periodontal inflammation and bone loss in wild-type mice. Although LIP-subjected C3-deficient mice are protected relative to wild-type littermates and exhibit only modest bone loss, mini-FH strikingly inhibited bone loss even in C3-deficient mice. However, mini-FH failed to inhibit ligature-induced bone loss in mice doubly deficient in C3 and CD11b. These findings indicate that mini-FH can inhibit experimental periodontitis even in a manner that is independent of its complement regulatory activity and is mediated by complement receptor 3 (CD11b/CD18). Consistent with this notion, a complement receptor 3-interacting recombinant FH segment that lacks complement regulatory activity (specifically encompassing SCRs 19 and 20; FH19-20) was also able to suppress bone loss in LIP-subjected C3-deficient mice. In conclusion, mini-FH appears to be a promising candidate therapeutic for periodontitis by virtue of its ability to suppress bone loss via mechanisms that both include and go beyond its complement regulatory activity.

Abstract A023: Structural basis for regulation of MAPK signaling by DUSP5 and DUSP6

Abstract The Ras/mitogen-activated protein kinase (MAPK) cascade plays an essential role in several critical cellular processes, such as cell growth and proliferation, and hyperactivation of this pathway is frequently observed in cancer. DUSP5 and DUSP6 are dual specificity phosphatases (capable of dephosphorylating phospho-tyrosines as well as phospho-serines/threonines) that act as key regulators of MAPK signaling that inactivate ERK by dephosphorylating its activation loop and sequestering ERK in an inactive state. DUSP5 and DUSP6 have been reported to function as tumor suppressors in some Ras-driven cancers, including skin cancer and pancreatic cancer, while promoting tumorigenesis in other contexts, such as thyroid cancer or glioblastoma. Despite the importance of these phosphatases in modulating ERK signaling, the structural basis for their interaction with ERK and their dual specificity remain poorly understood. It has also been reported that ERK binding allosterically activates DUSP6, but not DUSP5, and the mechanism underlying this is unclear. While structures of individual domains of these proteins have been solved, there are no structures of full-length DUSP5 or DUSP6, or of an ERK/DUSP complex. To gain insight into the molecular determinants of ERK dephosphorylation by these phosphatases, we have purified full-length DUSP5 and DUSP6, as well as the phosphatase domains of both proteins. We have also generated full-length phosphorylated ERK2 (pERK2). Through in vitro dephosphorylation assays, we have observed that full-length DUSP5 and DUSP6 dephosphorylate ERK2 much more rapidly than the isolated phosphatase domains, indicating an essential role for their N-terminal regulatory domains in this process. Using biolayer interferometry, we show that catalytically inactive mutants of full-length DUSP5 and DUSP6 bind very tightly to pERK2 with low nanomolar affinity. We have reconstituted complexes of pERK2 bound to catalytically inactive full-length DUSP5 or DUSP6, and we are pursuing structures of these complexes using x-ray crystallography and cryo-electron microscopy. These studies will improve our understanding of the regulation of ERK and its phosphatases DUSP5 and DUSP6 and shed light on the similarities and differences between these complexes. These structures could also potentially reveal the structural features underlying the dual specificity of DUSP5 and DUSP6. Citation Format: Jennifer E. Kung, Jawahar Sudhamsu. Structural basis for regulation of MAPK signaling by DUSP5 and DUSP6 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A023.

Novel function for gluconeogenic enzyme, FBP1, in T cell activation and expansion

Abstract During an immune response, naïve T lymphocytes undergo rapid clonal expansion and must reprogram their metabolism to meet high energetic and biosynthetic demands. The sudden increase in metabolic activity associated with a proliferative burst generates reactive oxygen species (ROS) increasing intracellular oxidative stress, which can disrupt homeostasis or trigger cell death, unless properly regulated. Our studies show that a constitutively active isoform of gluconeogenic enzyme, fructose 1,6-bisphosphatase 1 (FBP1), facilitates re-entry of glucose carbons into the pentose phosphate pathway to ensure the sustained production of NADPH for redox homeostasis and lipid synthesis in stimulated T cells as they transition to a phase of rapid growth, and is essential for their unperturbed clonal expansion and for an effective immune response. Wildtype (wt) FBP1, which catalyzes hydrolysis of fructose-1,6 bisphosphate in an irreversible reaction, has a C-terminal catalytic domain and an inhibitory N-terminal regulatory domain. We demonstrate that a short isoform of FBP1 representing the catalytic domain is induced in human T cells within 24 hours of co-stimulation. The short isoform, FBP1-CD, is the product of an alternatively spliced transcript and is resistant to inhibitors that suppress activity of wt FBP1, which harbors the regulatory domain. FBP1-CD levels and enzyme activity are significantly reduced 5 days post-stimulation, correlating positively with proliferation and inversely with ROS and apoptosis in T cells. In addition to new insights into immune regulation, our studies reveal a broader role for FBP1 in regulating proliferative metabolism and offer a rational explanation for the suppression of FBP1 in solid tumors. Supported by grant from NIH (R21 AI168777)

The role of class IIa histone deacetylases in regulating endothelial function.

Vascular endothelial cells (ECs) are monolayer cells located in the inner layer of the blood vessel. Endothelial function is crucial in maintaining local and systemic homeostasis and is precisely regulated by sophisticated signaling pathways and epigenetic regulation. Endothelial dysfunctions are the main factors for the pathophysiological process of cardiovascular and cerebrovascular diseases like atherosclerosis, hypertension, and stroke. In these pathologic processes, histone deacetylases (HDACs) involve in epigenetic regulation by removing acetyl groups from lysine residues of histones and regulating downstream gene expression. Among all HDACs, Class IIa HDACs (HDAC4, 5, 7, 9) contain only an N-terminal regulatory domain, exert limited HDAC activity, and present tissue-specific gene regulation. Here, we discuss and summarize the current understanding of this distinct subfamily of HDACs in endothelial cell functions (such as angiogenesis and immune response) with their molecular underpinnings. Furthermore, we also present new thoughts for further investigation of HDAC inhibitors as a potential treatment in several vascular diseases.

Investigating mutant hydrophobic patch exposure in troponin C using molecular dynamics-based free energy methods.

Contraction of cardiomyocytes is orchestrated through calcium-binding to the N-terminal regulatory domain of cardiac troponin C (NcTnC). This binding event initiates a series of dynamic and conformational changes that expose a hydrophobic patch to which troponin I (TnI) associates. Cardiomyopathies have been linked to mutations in NcTnC that disrupt these precise interactions and modulate free energy of opening. Mutations that lower the free energy of opening have a calcium-sensitizing effect and those that increase free energy of opening have a calcium-desensitizing effect, like mutations implicated with hypertrophic (HCM) and dilated cardiomyopathy (DCM), respectively. Albeit mutations occurring in the loop region of NcTnC have been most extensively studied, information regarding mutations within the hydrophobic patch is lacking. Simulating the closed-to-open transition in NcTnC is challenging and typically requires microsecond timescales just to observe partial opening of the patch. Here, we use targeted molecular dynamics (TMD) to efficiently sample the landscape between the open and closed conformations. Umbrella sampling (US) was employed to predict the free energy of opening by using frames sourced from TMD, and preliminary data corroborates the expected trends for known HCM and DCM mutations in NcTnC. RosettaDesign was used to prepare and score novel mutation candidates by systematically mutating residues at eight different positions in the patch to almost every other amino acid. Based on the ref15_cart scoring function, up to three mutation candidates from each position were selected to proceed to further evaluation with US. In this work, we identify several mutations that are expected to perturb patch opening and present an innovative approach to quantitatively study the influence hydrophobic patch residues play in the molecular basis of myocyte contraction.

Myosin binding protein h dominates the "c-zone" in ultrafast swimming muscles of developing zebrafish.

The ‘C-zone’ of mature vertebrate skeletal muscle sarcomeres is characterized by the presence of Myosin-Binding Protein C (MyBP-C), which ‘tunes’ contractility via its N-terminal domain interactions with the myosin head region and/or the thin filament. The ultrafast swimming muscles of zebrafish larvae are a perfect model system to define MyBP-C isoform function, due to tractable genetics and easily characterized muscle mechanics. However, liquid chromatography mass spectrometry shows 5-day-old larval muscle expresses MyBP-C at only ∼5% of the ∼1:10 molar ratio with myosin heavy chain (MYH) typically associated with full C-zone occupancy, while a little-studied MyBP-C paralog, Myosin-Binding Protein H (MyBP-H) comprises the remaining ∼95%. MyBP-H is homologous to MyBP-C's C terminus, which binds to the myosin thick filament backbone, but it lacks analagous N-terminal regulatory domains, which are critical to MyBP-C's slowing of muscle shortening. Therefore, the presence of MyBP-H may underlie the extreme contractile speed of larval muscle (>30 lengths/s), and/or indicate its involvement in muscle development. To define MyBP-H's potential roles, we first imaged its sarcomeric localization in larvae expressing transgenic, FLAG-tagged MyBP-H. Immunofluorescence revealed characteristic fluorescence doublets at the sarcomere center, indicating MyBP-H binds throughout the C-zone. Interestingly, MyBP-H-null mutants engineered using CRISPR/Cas9 appear to develop normally, suggesting the developmental impact of MyBP-H may be subtle. Preliminary mechanical studies of MyBP-H-null larval muscles showed no change in twitch kinetics, tetanic force, or the Force:Velocity relationship, which might be expected, given MyBP-C content increases only minimally (From ∼5% to ∼10% of full C-zone occupancy). Although the role of MyBP-H in development remains an enigma, physiologically stressing the larvae may reveal MyBP-H's function. At a minimum, the MyBP-H null larvae provide a clean slate to repopulate the C-zone using transgenically expressed human or disease-causing MyBP-C isoforms.

The bacteriophage lambda integrase catalytic domain can be modified to act with the regulatory domain as a recombination-competent binary recombinase

Site-specific recombinase Int mediates integration of the bacteriophage λ genome into the E. coli chromosome. Integration occurs once the Int tetramer, assisted by the integration host factor IHF, forms the intasome, a higher order structure, within which Int, a heterobivalent protein, interacts with two non-homologous DNA sequences: the core recombination sites and the accessory arm sites. The binding to these sites is mediated by the catalytic C-terminal domain (CTD) and the regulatory N-terminal domain (NTD) of Int, respectively. Within Int, the NTD can activate or inhibit the recombination activity of the CTD depending on whether the NTD is bound to the arm sites. The CTD alone cannot mediate recombination, and even when the NTD and the CTD are mixed together as individual polypeptides, the NTD cannot trigger recombination in the CTD. In this work, we set to determine what modifications can unlock the recombination activity in the CTD alone and how the CTD can be modified to respond to recombination-triggering signals from the NTD. For this, we performed a series of genetic analyses which showed that a single mutation that stabilizes the CTD on DNA, E174K, allows the CTD to recombine the core DNA sequences. When the NTD is paired with the CTD(E174K) that also bears a short polypeptide from the C-terminus of the NTD, the resulting binary Int can recombine arm-bearing substrates. Our results provide insights into the molecular basis of the regulation of the Int activity and suggest how binary recombinases of the integrase type can be engineered.