Amino Acids In Loop Research Articles

Molecular Mechanism of pH-Induced Protrusion Configuration Switching in Piscine Betanodavirus Implies a Novel Antiviral Strategy.

Many viruses contain surface spikes or protrusions that are essential for virus entry. These surface structures can thereby be targeted by antiviral drugs to treat viral infections. Nervous necrosis virus (NNV), a simple nonenveloped virus in the genus of betanodavirus, infects fish and damages aquaculture worldwide. NNV has 60 conspicuous surface protrusions, each comprising three protrusion domains (P-domain) of its capsid protein. NNV uses protrusions to bind to common receptors of sialic acids on the host cell surface to initiate its entry via the endocytic pathway. However, structural alterations of NNV in response to acidic conditions encountered during this pathway remain unknown, while detailed interactions of protrusions with receptors are unclear. Here, we used cryo-EM to discover that Grouper NNV protrusions undergo low-pH-induced compaction and resting. NMR and molecular dynamics (MD) simulations were employed to probe the atomic details. A solution structure of the P-domain at pH 7.0 revealed a long flexible loop (amino acids 311-330) and a pocket outlined by this loop. Molecular docking analysis showed that the N-terminal moiety of sialic acid inserted into this pocket to interact with conserved residues inside. MD simulations demonstrated that part of this loop converted to a β-strand under acidic conditions, allowing for P-domain trimerization and compaction. Additionally, a low-pH-favored conformation is attained for the linker connecting the P-domain to the NNV shell, conferring resting protrusions. Our findings uncover novel pH-dependent conformational switching mechanisms underlying NNV protrusion dynamics potentially utilized for facilitating NNV entry, providing new structural insights into complex NNV-host interactions with the identification of putative druggable hotspots on the protrusion.

TALE gene family: identification, evolutionary and expression analysis under various exogenous hormones and waterlogging stress in Cucumis sativus L.

BackgroundThree Amino acid Loop Extension (TALE) belongs to the homeobox group of genes that are important constituents of plant systems. The TALE gene family is instrumental not only in growth and development but also plays an essential role in regulating plant response to environmental adversaries.ResultsIn the present study, we isolated 21 CsTALE genes from the cucumber (Cucumis sativus L.) genome database. Bioinformatics tools were put in place to understand the structural and functional components of the CsTALE gene family. The evolutionary analysis dissected them into seven subclades (KNOX-I, KNOX-II, and BELL-I to BELL-V). The cis-acting elements in the promoter region of CsTALE genes disclosed that they are key regulators of hormonal and stress-related processes. Additionally, the STRING database advocated the concerting role of CsTALE proteins with other key transcription factors potent in plant developmental biology. The CsmiR319 and CsmiR167a-3p targeting the CsTALE15 and CsTALE16, respectively, further assert the importance of the CsTALE gene family posttranscriptional-related processes. Tissue-specific gene expression unfolded the fundamental involvement of CsTALE genes as they were expressed throughout the developmental stages. Under waterlogging stress, the CsTALE17 expressed significantly higher values in WL, WL-NAA, and WL-ETH but not in WL-MeJA-treated samples.ConclusionsThe present study reveals the evolution and functions of the CsTALE gene family in cucumber. Our work will provide a platform that will help future researchers address the issue of waterlogging stress in the Yangtze River Delta.

Differential G protein activation by the long and short isoforms of the dopamine D2 receptor.

The dopamine D2 receptor is expressed as a short (D2S) and a long (D2L) isoform with 29 additional amino acids in the third intracellular loop. The D2S isoform shows higher presynaptic expression than the D2L isoform, and decreased D2S expression has recently been linked to an increased risk for schizophrenia. Here, we present the first investigation, at receptor isoform level, of kinetic differences in the G protein activation profiles of the D2S, compared with the D2L isoform. We employed a NanoBRET-based approach to G protein dissociation to interrogate the time-resolved coupling profile of 3×HA-tagged D2L and D2S to Gαi/o/z proteins in vitro. Using dopamine as a D2 receptor agonist, we observed a more pronounced activation of Gαo and Gαz than Gαi proteins by D2L compared with D2S. This differentiation was not observed for D2S, which activated Gαo and Gαz with lower efficacy than D2L. These signalling differences were preserved on second messenger level and were not due to differences in receptor expression. Expanding to a set of seven full and partial D2 receptor agonists showed these effects were not restricted to dopamine but rather a mutual, receptor-associated property. Contrasting this trend, we found that D2S activated G proteins faster than D2L upon full receptor activation. The findings highlight that both D2L and D2S are mechanistically able to activate all non-visual Gαi/o proteins. Thereby, they add to previous reports about isoform-specificity to certain Gαi/o proteins observed in specific cell types.

Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans

For canine parvovirus -2 (CPV-2), a zoonotic virus capable of cross-species transmission in animals, the amino acid changes of capsid protein VP2 are key factors when binding to other species' transferrin receptors (TfR). CPV-2 variants can spread from felines and canines, for example, to Carnivora, Artiodactyla, and Pholidota species, and CPV-2c variants are essential to spread from Carnivora to Artiodactyla and Pholidota species in particular. In our study, a CPV-2a variant maintained a relatively stable trend, and the proportion of CPV-2c gradually rose from 1980 to 2021. The VP2 amino acid sequence analysis showed that five amino acid mutations at 426E/D, 305H/D, and 297S may be necessary for the virus to bind to different host receptors. Meanwhile, receptor-binding loop regions and amino acid sites 87 L, 93 N, 232I, and 305Y were associated with CPV-2 cross-species transmission. The homology of TfRs in different hosts infected with CPV-2 ranged from 77.2 % to 99.0 %, and from pig to feline, canine, and humans was 80.7 %, 80.4 %, and 77.2 %, respectively. The amino acid residues of TfRs involved in the viral binding in those hosts are highly conserved, which suggests that CPV-2 may be capable of pig-to-human transmission. Our analysis of the origin, evolutionary trend, cross-species transmission dynamics, and genetic characteristics of CPV-2 when binding to host receptors provides a theoretical basis for further research on CPV-2's mechanism of cross-species transmission and for establishing an early warning and monitoring mechanism for the possible threat of CPV-2 to animal–human public security.

Epitope(s) involving amino acids of the fusion loop of Japanese encephalitis virus envelope protein is(are) important to elicit protective immunity.

Introduction of mutations into the fusion loop is one potential strategy for generating safe dengue and Zika vaccines by reducing the risk of severe dengue following subsequent infections, and for constructing live-attenuated vaccine candidates against newly emerging Japanese encephalitis virus (JEV) or Japanese encephalitis (JE) serocomplex virus. The monoclonal antibody studies indicated the fusion loop of JE serocomplex viruses primarily comprised non-neutralizing epitopes. However, the present study demonstrates that the JEV fusion loop plays a critical role in eliciting protective immunity in mice. Modifications to the fusion loop of JE serocomplex viruses might negatively affect vaccine efficacy compared to dengue and zika serocomplex viruses. Further studies are required to assess the impact of mutant fusion loop encoded by commonly used JEV vaccine strains on vaccine efficacy or safety after subsequent dengue virus infection.

Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel’s voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

PBX1: a TALE of two seasons-key roles during development and in cancer.

Pre-B cell leukemia factor 1 (PBX1) is a Three Aminoacid Loop Extension (TALE) homeodomain-containing transcription factor playing crucial roles in organ pattering during embryogenesis, through the formation of nuclear complexes with other TALE class and/or homeobox proteins to regulate target genes. Its contribution to the development of several organs has been elucidated mainly through the study of murine knockout models. A crucial role for human development has been recently highlighted through the discovery of different de novo pathogenic PBX1 variants in children affected by developmental defects. In the adult, PBX1 is expressed in selected tissues such as in the brain, in the gastro-intestinal and urinary systems, or in hematopoietic stem and progenitor cells, while in other organs is barely detectable. When involved in the t(1;19) chromosomal translocation it acts as an oncogene, since the resulting fusion protein drives pre-B cell leukemia, due to the induction of target genes not normally targeted by the native protein. Its aberrant expression has been associated to tumor development, progression, or therapy-resistance as in breast cancer, ovarian cancer or myeloproliferative neoplasm (MPN). On the other hand, in colorectal cancer PBX1 functions as a tumor suppressor, highlighting its context-dependent role. We here discuss differences and analogies of PBX1 roles during embryonic development and in cancer, focusing mainly on the most recent discoveries.

Rational design of a cross-type HPV vaccine through immunodominance shift guided by a cross-neutralizing antibody

In vaccine development, broadly or cross-type neutralizing antibodies (bnAbs or cnAbs) are frequently targeted to enhance protection. Utilizing immunodominant antibodies could help fine-tune vaccine immunogenicity and augment the precision of immunization strategies. However, the methodologies to capitalize on the attributes of bnAbs in vaccine design have not been clearly elucidated. In this study, we discovered a cross-type neutralizing monoclonal antibody, 13H5, against human papillomavirus 6 (HPV6) and HPV11. This nAb exhibited a marked preference for HPV6, demonstrating superior binding activity to virus-like particles (VLPs) and significantly higher prevalence in anti-HPV6 human serum as compared to HPV11 antiserum (90% vs. 31%). Through co-crystal structural analysis of the HPV6 L1 pentamer:13H5 complex, we delineated the epitope as spanning four segments of amino acids (Phe42-Ala47, Gly172-Asp173, Glu255-Val275, and Val337-Tyr351) on the L1 surface loops. Further interaction analysis and site-directed mutagenesis revealed that the Ser341 residue in the HPV6 HI loop plays a critical role in the interaction between 13H5 and L1. Substituting Ser341 with alanine, which is the residue type present in HPV11 L1, almost completely abolished binding activity to 13H5. By swapping amino acids in the HPV11 HI loop with corresponding residues in HPV6 L1 (Ser341, Thr338, and Thr339), we engineered chimeric HPV11-6HI VLPs. Remarkably, the chimeric HPV11-6HI VLPs shifted the high immunodominance of 13H5 from HPV6 to the engineered VLPs and yielded comparable neutralization titers for both HPV6 and HPV11 in mice and non-human primates. This approach paves the way for the design of broadly protective vaccines from antibodies within the main immunization reservoir.

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. I. Effect of Solvent Viscosity and Macromolecular Crowding.

Protein/solvent interactions largely influence protein dynamics, particularly motions in unfolded and intrinsically disordered proteins (IDPs). Here, we apply triplet-triplet energy transfer (TTET) to investigate the coupling of internal protein motions to solvent motions by determining the effect of solvent viscosity (η) and macromolecular crowding on the rate constants of loop formation (kc) in several unfolded polypeptide chains including IDPs. The results show that the viscosity dependence of loop formation depends on amino acid sequence, loop length, and co-solute size. Below a critical size (rc), co-solutes exert a maximum effect, indicating that under these conditions microviscosity experienced by chain motions matches macroviscosity of the solvent. rc depends on chain stiffness and reflects the length scale of the chain motions, i.e., it is related to the persistence length. Above rc, the effect of solvent viscosity decreases with increasing co-solute size. For co-solutes typically used to mimic cellular environments, a scaling of kc ∝ η-0.1 is observed, suggesting that dynamics in unfolded proteins are only marginally modulated in cells. The effect of solvent viscosity on kc in the small co-solute limit (below rc) increases with increasing chain length and chain flexibility. Formation of long and very flexible loops exhibits a kc ∝ η-1 viscosity dependence, indicating full solvent coupling. Shorter and less flexible loops show weaker solvent coupling with values as low as kc ∝ η-0.75±0.02. Coupling of formation of short loops to solvent motions is very little affected by amino acid sequence, but solvent coupling of long-range loop formation is decreased by side chain sterics.

Dynamic regulation of the serine loop by distant mutations reveals allostery in cryptochrome1

Cryptochromes (CRYs) are essential components of the molecular clock that generates circadian rhythm. They inhibit BMAL1/CLOCK-driven transcription at the molecular level. There are two CRYs that have differential functions in the circadian clock in mammals. It is not precisely known how they achieve such differential functions. In this study, we performed molecular dynamic simulations on eight CRY mutants that have been experimentally shown to exhibit reduced repressor activities. Our results revealed that mutations in CRY1 affect the dynamic behavior of the serine loop and the availability of the secondary pocket, but not in CRY2. Further analysis of these CRY1 mutants indicated that the differential flexibility of the serine loop leads to changes in the volume of the secondary pocket. We also investigated the weak interactions between the amino acids in the serine loop and those in close proximity. Our findings highlighted the crucial roles of S44 and S45 in the dynamic behavior of the serine loop, specifically through their interactions with E382 in CRY1. Considering the clinical implications of altered CRY1 function, our study opens up new possibilities for the development of drugs that target the allosteric regulation of CRY1. Communicated by Ramaswamy H. Sarma

Three-amino acid loop extension homeodomain proteins regulate stress responses and encystation in Entamoeba.

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein-encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST-EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down-regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation-induced genes.

Outer membrane protein OMP76 of Riemerella anatipestifer contributes to complement evasion and virulence by binding to duck complement factor vitronectin

ABSTRACT Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer. The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.

Bioengineered Bovine Papillomavirus L1 Protein Virus-like Particle (VLP) Vaccines for Enhanced Induction of CD8 T Cell Responses through Cross-Priming.

Safe and effective T cell vaccines are needed for the treatment or prevention of cancers as well as infectious agents where vaccines for neutralizing antibodies have performed poorly. Recent research highlights an important role for tissue-resident memory T cells (TRM cells) in protective immunity and the role of a subset of dendritic cells that are capable of cross-priming for the induction of TRM cells. However, efficient vaccine technologies that operate through cross-priming and induce robust CD8+ T cell responses are lacking. We developed a platform technology by genetically engineering the bovine papillomavirus L1 major capsid protein to insert a polyglutamic acid/cysteine motif in place of wild-type amino acids in the HI loop. Virus-like particles (VLPs) are formed by self-assembly in insect cells infected with a recombinant baculovirus. Polyarginine/cysteine-tagged antigens are linked to the VLP by a reversible disulfide bond. The VLP possesses self-adjuvanting properties due to the immunostimulatory activity of papillomavirus VLPs. Polyionic VLP vaccines induce robust CD8+ T cell responses in peripheral blood and tumor tissues. A prostate cancer polyionic VLP vaccine was more efficacious than other vaccines and immunotherapies for the treatment of prostate cancer in a physiologically relevant murine model and successfully treated more advanced diseases than the less efficacious technologies. The immunogenicity of polyionic VLP vaccines is dependent on particle size, reversible linkage of the antigen to the VLP, and an interferon type 1 and Toll-like receptor (TLR)3/7-dependent mechanism.

Research progress of PBX1 in developmental and regenerative medicine.

Pre-B-cell leukemia transcription factor 1 (PBX1) proteins are a subfamily of evolutionarily conserved atypical homeodomain transcription factors belonging to the superfamily of triple amino acid loop extension homeodomain proteins. PBX family members play crucial roles in the regulation of various pathophysiological processes. This article reviews the research progress on PBX1 in terms of structure, developmental function, and regenerative medicine. The potential mechanisms of development and research targets in regenerative medicine are also summarized. It also suggests a possible link between PBX1 in the two domains, which is expected to open up a new field for future exploration of cell homeostasis, as well as the regulation of endogenous danger signals. This would provide a new target for the study of diseases in various systems.

AXL-Receptor Targeted 14FN3 Based Single Domain Proteins (Pronectins™) from 3 Synthetic Human Libraries as Components for Exploring Novel Bispecific Constructs against Solid Tumors.

A highly specific AXL-receptor targeted family of non-immunoglobulin, single domain protein binders (Pronectins™) have been isolated from three (3) synthetic libraries that employ the human scaffold of the 14th domain of Fibronectin III (14FN3) and evolutionary CDRs diversity of over 25 billion loop sequences. The three libraries, each containing diversity in two loops, were designed to expand upon a human database of more than 6000 natural scaffold sequences and approximately 3000 human loop sequences. We used a bioinformatic-based approach to maximize "human" amino acid loop diversity and minimize or prevent altogether CDR immunogenicity created by the use of mutagenesis processes to generate diversity. A combination of phage display and yeast display was used to isolate 59 AXL receptor targeted Pronectins with KD ranging between 2 and 100 nM. FACS analysis with tumor cells over-expressing AXL and the use of an AXL knock-out cell line allowed us to identify Pronectin candidates with exquisite specificity for AXL receptor. Based upon several in vitro cell-based tests, we selected the best candidate, AXL54, to further characterize its in vitro cancer cells killing activity. Finally, AXL54 was used to produce the first bi-specific T cell engager protein (AXL54 [Pronectin]-linker-scFV CD3), a "new in class" protein for further testing of its anti-tumor activity in vitro and in vivo.

PMON170 Treating human primary non-functioning pituitary adenoma cells with cabergoline

Abstract Clinically non-functioning pituitary adenomas (NFPAs) represent the second most common subtype of pituitary tumours, representing 15-43% of all pituitary adenomas. NFPAs are usually benign tumours with no clinical evidence of hormonal hypersecretion. In most of the cases the tumours are arising from the gonadotroph linage, but silent corticotroph, somatotroph, thyrotroph and rarely lactotroph cell characteristics can be discovered with immunostaining. As a result of lack of hormonal hyper-secretion, the diagnosis of NFPAs is made most often when the patient presents with mass effects due to a macroadenoma. NFPAs are most common tumour types in surgical series, and their primary treatment is indeed surgery. However, complete resection is achieved only in about 66% of the cases, and 20% of gross total resected tumours recur after 10 years. Over the past few decades, remarkable progress has been achieved regarding medical treatment options for pituitary tumours. However, NFPAs remain the only subtype with no widely accepted pharmacological treatment. Dopamine receptor type 2 (DRD2) and somatostatin receptor (SST) expression have been demonstrated in NFPAs, prompting the investigation of dopamine agonists and somatostatin analogues as potential treatment strategies. The DRD2 agonist cabergoline is already used as first-line treatment of prolactinomas to induce tumour shrinkage and reduce prolactin secretion. Here we aim to investigate the efficacy of cabergoline on human NFPA tissue. We assessed DRD2 expression levels via immunohistochemistry and qPCR in a large cohort of NFPAs (n=40) and in few prolactinomas (n=10). D2DR has two different isoforms, long (D2RL) and short (D2RS), which are differently expressed and can induce different effects on cell viability. In cabergoline-sensitive prolactinomas D2RS is the predominant isoform. NFPAs shown 10-fold and 5-fold decrease expression of D2RS and D2RL compared to prolactinomas, respectively. Additionally, in NFPAs we observed a significant decrease of cAMP production after cabergoline treatment (45% ±7; p<0.0001); however, viability after one-week cabergoline treatment showed only 4% (±0.7; p<0.0002) reduction, compared to a 10% decrease in prolactinomas (p<0.05). We also observed no difference in secretion of chromogranin A release in NFPAs upon treatment with cabergoline. We have also seen in NFPAs downregulation of Ga i2 protein expression levels, highlighting the general decrease of expression levels in the dopamine pathway (50% less, p<0.004). Our data suggest that the difference in cabergoline responses between NFPAs and prolactinomas may be due to their distinct expression of D2DR isoforms, which differ by 29 amino acids in the third cytoplasmic loop, essential for G-protein binding. It has been shown that D2RL, but not D2RS, requires Gai2. The difference in D2R isoform expression could translate to distinct G-protein activation, ultimately leading to the contrasting viability results after cabergoline treatment. Taken together, these data will help inform future treatment strategies for patients with NFPAs. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Minimal structure of IRAK-1 to induce degradation of TRAF6

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1–102), a serine/proline/threonine-rich ProST domain (amino acids 103–197), a central kinase domain with an activation loop (amino acids 198–522), and the C-terminal C1 and C2 domains (amino acids 523–712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585–591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants–including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

Gold-Mediated Multiple Cysteine Arylation for the Construction of Highly Constrained Bicycle Peptides.

Bicycles are constrained bicyclic peptides formed through reaction of three cysteine residues within a linear sequence with a trivalent, symmetrical small molecule scaffold. Bicycles with high binding affinities to therapeutically important targets can be discovered using screening technologies such as phage display. Increasing the chemical diversity of Bicycles should improve the probability of finding hits to new targets and can be achieved by expanding the toolbox of Bicycle forming chemistries. Gold(III) S-arylation has recently been described as a method for the efficient bioconjugation of cysteine residues under conditions compatible with phage display. Herein, we explore the scope and generality of this methodology for Bicycle construction through the synthesis and evaluation of four novel tris-Gold complexes. These new scaffolds were systematically reacted with a variety of peptide sequences, varying in amino acid loop lengths. All four scaffolds proved to be capable and selective reactive partners for each peptide sequence and afforded the desired Bicycle products in 13-48% isolated yield. This work exemplifies Gold-mediated arylation as a general approach for construction of novel, highly constrained Bicycles.

The TALE never ends: A comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects.

PBX1 is a highly conserved atypical homeodomain transcription factor (TF) belonging to the TALE (three amino acid loop extension) family. Dimerized with other TALE proteins, it can interact with numerous partners and reach dozens of regulating sequences, suggesting its role as a pioneer factor. PBX1 is expressed throughout the embryonic stages (as early as the blastula stage) in vertebrates. In human, PBX1 germline variations are linked to syndromic renal anomalies (CAKUTHED). In this review, we summarized available data on PBX1 functions, PBX1-deficient animal models, and PBX1 germline variations in humans. Two types of genetic alterations were identified in PBX1 gene. PBX1 missense variations generate a severe phenotype including lung hypoplasia, cardiac malformations, and sexual development defects (DSDs). Conversely, truncating variants generate milder phenotypes (mainly cryptorchidism and deafness). We suggest that defects in PBX1 interactions with various partners, including proteins from the HOX (HOXA7, HOXA10, etc.), WNT (WNT9B, WNT3), and Polycomb (BMI1, EED) families are responsible for abnormal proliferation and differentiation of the embryonic mesenchyme. These alterations could explain most of the defects observed in humans. However, some phenotype variability (especially DSDs) remains poorly understood. Further studies are needed to explore the TALE family in greater depth.

A coiled-coil protein associates Polycomb Repressive Complex 2 with KNOX/BELL transcription factors to maintain silencing of cell differentiation-promoting genes in the shoot apex.

Polycomb repressive complex 2 (PRC2), which mediates the deposition of H3K27me3 histone marks, is important for developmental decisions in animals and plants. In the shoot apical meristem (SAM), Three Amino acid Loop Extension family KNOTTED-LIKE HOMEOBOX /BEL-like (KNOX/BELL) transcription factors are key regulators of meristem cell pluripotency and differentiation. Here, we identified a PRC2-associated coiled-coil protein (PACP) that interacts with KNOX/BELL transcription factors in rice (Oryza sativa) shoot apex cells. A loss-of-function mutation of PACP resulted in differential gene expression similar to that observed in PRC2 gene knockdown plants, reduced H3K27me3 levels, and reduced genome-wide binding of the PRC2 core component EMF2b. The genomic binding of PACP displayed a similar distribution pattern to EMF2b, and genomic regions with high PACP- and EMF2b-binding signals were marked by high levels of H3K27me3. We show that PACP is required for the repression of cell differentiation-promoting genes targeted by a rice KNOX1 protein in the SAM. PACP is involved in the recruitment or stabilization of PRC2 to genes targeted by KNOX/BELL transcription factors to maintain H3K27me3 and gene repression in dividing cells of the shoot apex. Our results provide insight into PRC2-mediated maintenance of H3K27me3 and the mechanism by which KNOX/BELL proteins regulate SAM development.