Effector Binding Sites Research Articles

CRISPR/Cas9-generated mutations in a sugar transporter gene reduce cassava susceptibility to bacterial blight.

Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing the expression of host susceptibility (S) genes. Xanthomonas phaseoli pv. manihotis (Xpm) causes bacterial blight on the staple food crop cassava (Manihot esculenta Crantz). The Xpm effector TAL20 induces ectopic expression of the S gene Manihot esculenta Sugars Will Eventually be Exported Transporter 10a (MeSWEET10a), which encodes a sugar transporter that contributes to cassava bacterial blight (CBB) susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to the MeSWEET10a TAL20 effector binding site and/or coding sequence. In several of the regenerated lines, MeSWEET10a expression was no longer induced by Xpm, and in these cases, we observed reduced CBB disease symptoms post Xpm infection. Because MeSWEET10a is expressed in cassava flowers, we further characterized the reproductive capability of the MeSWEET10a promoter and coding sequence mutants. Lines were crossed to themselves and to wild-type plants. The results indicated that expression of MeSWEET10a in female, but not male, flowers is critical to produce viable F1 seed. In the case of promoter mutations that left the coding sequence intact, viable F1 progeny were recovered. Taken together, these results demonstrate that blocking MeSWEET10a induction is a viable strategy for decreasing cassava susceptibility to CBB and that ideal lines will contain promoter mutations that block TAL effector binding while leaving endogenous expression of MeSWEET10a unaltered.

Structures of the DarR transcription regulator reveal unique modes of second messenger and DNA binding

The mycobacterial repressor, DarR, a TetR family regulator (TFR), was the first transcription regulator shown to bind c-di-AMP. However, the molecular basis for this interaction and the mechanism involved in DNA binding by DarR remain unknown. Here we describe DarR-c-di-AMP and DarR-DNA structures and complementary biochemical assays. The DarR-c-di-AMP structure reveals a unique effector binding site for a TFR, located between DarR dimer subunits. Strikingly, we show this motif also binds cAMP. The location of the adenine nucleotide binding site between subunits suggests this interaction may facilitate dimerization and hence DNA binding. Indeed, biochemical assays show cAMP enhances DarR DNA binding. Finally, DarR-DNA structures reveal a distinct TFR DNA-binding mechanism involving two interacting dimers on the DNA. Thus, the combined data unveil a newly described second messenger binding motif and DNA binding mode for this important family of regulators.

Neuromodulators as Interdomain Signaling Molecules Capable of Occupying Effector Binding Sites in Bacterial Transcription Factors

Hormones and neurotransmitters are important components of inter-kingdom signaling systems that ensure the coexistence of eukaryotes with their microbial community. Their ability to affect bacterial physiology, metabolism, and gene expression was evidenced by various experimental approaches, but direct penetration into bacteria has only recently been reported. This opened the possibility of considering neuromodulators as potential effectors of bacterial ligand-dependent regulatory proteins. Here, we assessed the validity of this assumption for the neurotransmitters epinephrine, dopamine, and norepinephrine and two hormones (melatonin and serotonin). Using flexible molecular docking for transcription factors with ligand-dependent activity, we assessed the ability of neuromodulators to occupy their effector binding sites. For many transcription factors, including the global regulator of carbohydrate metabolism, CRP, and the key regulator of lactose assimilation, LacI, this ability was predicted based on the analysis of several 3D models. By occupying the ligand binding site, neuromodulators can sterically hinder the interaction of the target proteins with the natural effectors or even replace them. The data obtained suggest that the direct modulation of the activity of at least some bacterial transcriptional factors by neuromodulators is possible. Therefore, the natural hormonal background may be a factor that preadapts bacteria to the habitat through direct perception of host signaling molecules.

Structural basis of transcription factor YhaJ for DNT detection

Detection of landmines without harming personnel is a global issue. The bacterial transcription factor YhaJ selectively detects metabolites of explosives, and it can be used as a key component of DNT biosensors. However, the wild-type YhaJ has a binding affinity that is not sufficient for the detection of trace amounts of explosives leaked from landmines buried in the soil. Here, we report crystal structures of the effector-binding domain of YhaJ in both the apo- and effector-bound forms. A structural comparison of the two forms revealed that the loop above the primary effector-binding site significantly switches its conformation upon effector binding. The primary effector-binding site involves hydrophobic and polar interactions, having specificity to hydroxyl-substituted benzene compounds. The structures explain the mechanism of activity-enhancing mutations and provide information for the rational engineering of YhaJ biosensors for the sensitive detection of explosives.

Dynamic allostery in thrombin-a review.

Thrombin is a serine protease that catalyzes a large number of different reactions including proteolytic cleave of fibrinogen to make the fibrin clot (procoagulant activity), of the protease activated receptors (for cell signaling) and of protein C generating activated protein C (anticoagulant activity). Thrombin has an effector binding site called the anion binding exosite 1 that is allosterically coupled to the active site. In this review, we survey results from thermodynamic characterization of the allosteric coupling as well as hydrogen-deuterium exchange mass spectrometry to reveal which parts of the thrombin structure are changed upon effector binding and/or mutagenesis, and finally NMR spectroscopy to characterize the different timescales of motions elicited by the effectors. We also relate the experimental work to computational network analysis of the thrombin-thrombomodulin complex.

Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance

Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance. Pyruvate kinase (PYK) is the last enzyme in this pathway that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP) and plays a crucial role in controlling carbon flux; however, while S.pneumoniae PYK (SpPYK) is indispensable for growth, surprisingly little is known about its functional properties. Here, we report that compromising mutations in SpPYK confers resistance to the antibiotic fosfomycin, which inhibits the peptidoglycan synthesis enzyme MurA, implying a direct link between PYK and cell wall biogenesis. The crystal structures of SpPYK in the apo and ligand-bound states reveal key interactions that contribute to its conformational change as well as residues responsible for the recognition of PEP and the allosteric activator fructose 1,6-bisphosphate (FBP). Strikingly, FBP binding was observed at a location distinct from previously reported PYK effector binding sites. Furthermore, we show that SpPYK could be engineered to become more responsive to glucose 6-phosphate instead of FBP by sequence and structure-guided mutagenesis of the effector binding site. Together, our work sheds light on the regulatory mechanism of SpPYK and lays the groundwork for antibiotic development that targets this essential enzyme.

Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Bacterial NadQ (COG4111) is a Nudix-like, ATP-responsive regulator of NAD biosynthesis

Nicotinamide-adenine dinucleotide (NAD) is centrally important to metabolic reactions that involve redox chemistry. In bacteria, NAD biosynthesis is controlled by different transcription factors, depending on the species. Among the four regulators identified so far, the protein NadQ is reported to act as a repressor of the de novo NAD biosynthetic pathway in proteobacteria. Using comparative genomics, a systematic reconstruction of NadQ regulons in thousands of fully sequenced bacterial genomes has been performed, confirming that NadQ is present in α-proteobacteria and some β- and γ-proteobacteria, including pathogens like Bordetella pertussis and Neisseria meningitidis, where it likely controls de novo NAD biosynthesis. Through mobility shift assay and mutagenesis, the DNA binding activity of NadQ from Agrobacterium tumefaciens was experimentally validated and determined to be suppressed by ATP. The crystal structures of NadQ in native form and in complex with ATP were determined, indicating that NadQ is a dimer, with each monomer composed of an N-terminal Nudix domain hosting the effector binding site and a C-terminal winged helix-turn-helix domain that binds DNA. Within the dimer, we found one ATP molecule bound, at saturating concentration of the ligand, in keeping with an intrinsic asymmetry of the quaternary structure. Overall, this study provided the basis for depicting a working model of NadQ regulation mechanism.

Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

Using fungible biosensors to evolve improved alkaloid biosyntheses.

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC50) < 30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine. High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, a precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids.

Abstract 2918: Comprehensive survey of KRas4B membrane interaction and orientation

Abstract Three RAS genes encode four Ras proteins, HRas, NRas, and KRas splice variants, KRas4A and KRas4B. In human cancers, KRAS oncogene is highly expressed (85%) as compared to other RAS oncogenes, NRAS (11%) and HRAS (4%). Oncogenic Ras leads to aberrant cell proliferation signaling, primarily through the MAPK (Raf/MEK/ERK) pathway. At the plasma membrane, nanoclustered Ras proteins promote Raf activation, mediating the downstream signaling pathway. Among the Ras isoforms, KRas4B in the membrane has been studied extensively due to its higher level of mutations in cancer. Although a number of experimental studies have focused on KRas4B subcellular localization and their nanocluster formation at the signaling platform, the mechanistic details of the membrane anchorage and the membrane orientation of KRas4B at atomic resolution are still unknown. Here, comprehensive computational studies were performed for KRas4B at the anionic lipid bilayers, composed of the phospholipids, PS and PA, and the phosphoinositides, PIP2, PI4P, and PI5P. Wild-type and two oncogenic mutants, G12D and G12V KRas4B in the GTP- and GDP-bound states were considered. The lysine rich HVR was post-translationally modified with the farnesyl and methyl groups. Previous studies showed that wild-type KRas4B-GDP interacts weakly and nonspecifically with anionic phospholipids, while oncogenic mutations alter selective anionic lipid binding toward anionic lipids, PA, PI3P, PI4P, and PI5P. Our studies indicate that the membrane interaction of KRas4B is highly sensitive depending on the types of nucleotides, mutational states, and anionic lipids enriched the lipid bilayer. While the GTP-bound spontaneously inserts its prenyl group into the hydrophobic core of the bilayer, the occurrence of spontaneous insertion is reduced in the GDP-bound form. The distance of GTP-bound catalytic domain from the bilayer surface is longer than the GDP-bound form. The HVR is sequestered by the GDP-bound catalytic domain, which causes the catalytic domain to be occluded and increases the membrane interaction by the catalytic domain. In contrast, the GTP-bound catalytic domain releases its HVR and liberates from the membrane interaction. The occlusion of catalytic domain is less prevalent in the GTP-bound state. KRas4BG12D/G12V-GTP exhibits the active-state orientation, exposing its effector binding site for recruiting the effectors. While PA and PIP2 support the active-state orientation, the PIP4 enriched bilayer prevents the spontaneous prenyl insertion resulting in the occlusion of catalytic domain. Our results underscore the importance of detailed structural mechanisms of KRas4B membrane interaction and orientation, elucidating vital mechanistic role of KRas4B, a key membrane-anchored protein in cancer. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I. Citation Format: Hyunbum Jang, Mingzhen Zhang, Vadim Gaponenko, Ruth Nussinov. Comprehensive survey of KRas4B membrane interaction and orientation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2918.

Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between “on” (GTP‐bound) and “off” (GDP‐bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP‐bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP‐bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD) of approximately 1 μM for the binding of these peptides to Gαs‐GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas‐mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs‐independent stimulation of adenylyl cyclase activity by forskolin. Protein‐protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi‐GTP in addition to Gαs‐GTP. In summary, we have identified a peptide inhibitor specific for Gαs‐GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically‐encoded probe or derivatized as a cell‐penetrating peptide to inhibit Gαs‐mediated cell signaling.

Time Evolution of the Millisecond Allosteric Activation of Imidazole Glycerol Phosphate Synthase.

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we combine extensive molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the substrate-free form to the active ternary complex. IGPS is a heterodimeric bienzyme complex whose HisH subunit is responsible for hydrolyzing glutamine and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with oxyanion hole formation, in line with the experimentally observed 4500-fold activity increase in glutamine hydrolysis. The allosteric activation is controlled by correlated time-evolving dynamic networks connecting the effector and substrate binding sites. This computational strategy tailored to describe millisecond events can be used to rationalize the effect of mutations on the allosteric regulation and guide IGPS engineering efforts.

A Xanthomonas transcription activator-like effector is trapped in nonhost plants for immunity

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight in rice, delivers transcription activator-like effector (TALE) proteins into host cells to activate susceptibility or resistance (R) genes that promote disease or immunity, respectively. Nonhost plants serve as potential reservoirs of R genes; consequently, nonhost R genes may trap TALEs to trigger an immune response. In this study, we screened 17 Xoo TALEs for their ability to induce a hypersensitive response (HR) in the nonhost plant Nicotiana benthamiana (Nb); only AvrXa10 elicited an HR when transiently expressed in Nb. The HR generated by AvrXa10 required both the central repeat region and the activation domain, suggesting a specific interaction between AvrXa10 and a potential R-like gene in nonhost plants. Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death, and the transient expression of AvrXa10 in Nb induced immune responses. Genes targeted by AvrXa10 in the Nb genome were identified by transcriptome profiling and prediction of effector binding sites. Using several approaches (in vivo reporter assays, electrophoretic mobility-shift assays, targeted designer TALEs, and on-spot gene silencing), we confirmed that AvrXa10 targets NbZnFP1, a C2H2-type zinc finger protein that resides in the nucleus. Functional analysis indicated that overexpression of NbZnFP1 and its rice orthologs triggered cell death in rice protoplasts. An NbZnFP1 ortholog was also identified in tomato and was specifically activated by AvrXa10. These results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 to promote plant immunity in Nb.

Crystal structure of a MarR family protein from the psychrophilic bacterium Paenisporosarcina sp. TG-14 in complex with a lipid-like molecule.

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Å resolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the β1 and β2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

Deciphering the Molecular Terms of Arp2/3 Allosteric Regulation from All-Atom Simulations and Dynamical Network Theory.

The Arp2/3 molecular machine stimulates the generation of branched actin networks at the cytosolic surface of cellular membranes. Arp2/3 is thus pivotal for cell motility and migration, and its aberrant function is implicated in cancer invasion and metastasis. Here, all-atom multi μs-long molecular dynamics simulations and dynamical NetWork Analysis (NWA) unprecedentedly disclose the molecular terms of Arp2/3 regulation (activation/inhibition) by positive/negative allosteric modulators. After identifying the crucial structural elements underlying Arp2/3's conformational transition toward its active actin-polymerization-competent state, we decrypt the activating signaling paths heading from the allosteric effector (ATP) binding sites to these pivotal regions, also elucidating how small-molecule inhibitors scramble this signal-exchange. As a result, while ATP-induced signaling triggers a harmonious conformational transition toward active Arp2/3, the inhibitors disturb these information channels, desynchronizing Arp2/3 functional movements, thus hindering its activation. Our outcomes supply a conceptual basis for devising small-molecule inhibitors to block infiltrative cancer migration.

Crystal structure details of Vibrio fischeri DarR and mutant DarR-M202I from LTTR family reveals their activation mechanism

DarR, a novel member of the LTTR family derived from Vibrio fischeri, activates transcription in response to d-Asp and regulates the overexpression of the racD genes encoding a putative aspartate racemase, RacD. Here, the crystal structure of full-length DarR and its mutant DarR-M202I were obtained by X-ray crystallography. According to the electron density map analysis of full-length DarR, the effector binding site of DarR is occupied by 2-Morpholinoethanesulfonic acid monohydrate (MES), which could interact with amino acids in the effector binding site and stabilize the effector binding site. Furthermore, we elaborated the structure of DarR-M202I, where methionine is replaced by isoleucine resulting in overexpression of the downstream operon. By comparing DarR-MES and DarR-M202I, we found similar behavior of DarR-MES in terms of the stability of the RD active pocket and the deflection angle of the DBD. The Isothermal titration calorimetry and Gel-filtration chromatography experiments showed that only when the target DNA sequence of a particular quasi-palindromic sequence exceeds 19 bp, DarR can effectively bind to racD promoter. This study will help enhance our understanding of the mechanism in the transcriptional regulation of LTTR family transcription factors.

Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum.

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.

Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A–SLP2-A interaction and RAB27A–MUNC13-4 interaction, but it does not affect the RAB27A–melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A–MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.

Effects of HTR1B 3\u2032 region polymorphisms and functional regions on gene expression regulation

BackgroundThe HTR1B gene encodes the 5-hydroxytryptamine (5-HT1B) receptor, which is involved in a variety of brain activities and mental disorders. The regulatory effects of non-coding regions on genomic DNA are one of many reasons for the cause of genetic-related diseases. Post-transcriptional regulation that depends on the function of 3′ regulatory regions plays a particularly important role. This study investigated the effects, on reporter gene expression, of several haplotypes of the HTR1B gene (rs6297, rs3827804, rs140792648, rs9361234, rs76194807, rs58138557, and rs13212041) and truncated fragments in order to analyze the function of the 3′ region of HTR1B.ResultsWe found that the haplotype, A-G-Del-C-T-Ins-A, enhanced the expression level compared to the main haplotype; A-G-Del-C-G-Ins-A; G-G-Del-C-G-Ins-G decreased the expression level. Two alleles, rs76194807T and rs6297G, exhibited different relative luciferase intensities compared to their counterparts at each locus. We also found that + 2440 ~ + 2769 bp and + 1953 ~ + 2311 bp regions both had negative effects on gene expression.ConclusionsThe 3′ region of HTR1B has a regulatory effect on gene expression, which is likely closely associated with the interpretation of HTR1B-related disorders. In addition, the HTR1B 3′ region includes several effector binding sites that induce an inhibitory effect on gene expression.