Identifying challenges and opportunities for histone peptides in protein engineering platforms

Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-β family

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-β) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-β family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

RHAMM-Target Peptides as Molecular Imaging Probes for the Imaging of RHAMM-Expressing Cancer Cells

The incidence of cancer in the world is growing steadily. Therefore, it is necessary to develop new approaches for the early diagnosis of cancer. This work is devoted to the study of the potential of RHAMM-target peptides for molecular diagnosis of cancer. The key amino acids of the RHAMM target peptides were identified by the alanine scan method. The specificity of binding of peptides to RHAMM-CT was assessed using competitive HA substitution by the ELISA method. RHAMM-CT was obtained by genetic engineering and isolated by affinity chromatography. The interaction of RHAMM target peptides with the surface receptor of tumor cells was evaluated by confocal microscopy. It has been shown that fragment EEGEEZ in the peptides' composition is necessary for binding to the RHAMM-CT. The results showed that the RHAMM-target peptides bind specifically to the RHAMM-CT and competitively substituted HA at the RHAMM. It has been found that aggrecan is unable to displace peptides from the HA binding site of RHAMM-CT. The results showed that the FITC peptide binds specifically to RHAMM on the surface of prostate cancer cells. Therefore, RHAMM-target peptides have the potential for early molecular diagnosis of cancer.

The influence of various regions of the FOXP2 sequence on its structure and DNA binding function

FOX proteins are a superfamily of transcription factors which share a DNA binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In this study we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

Funneled energy landscape unifies principles of protein binding and evolution

Most proteins have evolved to spontaneously fold into native structure and specifically bind with their partners for the purpose of fulfilling biological functions. According to Darwin, protein sequences evolve through random mutations, and only the fittest survives. The understanding of how the evolutionary selection sculpts the interaction patterns for both biomolecular folding and binding is still challenging. In this study, we incorporated the constraint of functional binding into the selection fitness based on the principle of minimal frustration for the underlying biomolecular interactions. Thermodynamic stability and kinetic accessibility were derived and quantified from a global funneled energy landscape that satisfies the requirements of both the folding into the stable structure and binding with the specific partner. The evolution proceeds via a bowl-like evolution energy landscape in the sequence space with a closed-ring attractor at the bottom. The sequence space is increasingly reduced until this ring attractor is reached. The molecular-interaction patterns responsible for folding and binding are identified from the evolved sequences, respectively. The residual positions participating in the interactions responsible for folding are highly conserved and maintain the hydrophobic core under additional evolutionary constraints of functional binding. The positions responsible for binding constitute a distributed network via coupling conservations that determine the specificity of binding with the partner. This work unifies the principles of protein binding and evolution under minimal frustration and sheds light on the evolutionary design of proteins for functions.

CD105 is a prognostic marker and valid endothelial target for microbubble platforms in cholangiocarcinoma

Purpose The current treatment outcomes in cholangiocarcinoma are poor with cure afforded only by surgical extirpation. The efficacy of targeting the tumoural endothelial marker CD105 in cholangiocarcinoma, as a basis for potential microbubble-based treatment, is unknown and was explored here. Methods Tissue expression of CD105 was quantified using immunohistochemistry in 54 perihilar cholangiocarcinoma samples from patients who underwent resection in a single centre over a ten-year period, and analysed against clinicopathological data. In vitro flow assays using microbubbles functionalised with CD105 antibody were conducted to ascertain specificity of binding to murine SVR endothelial cells. Finally, CD105-microbubbles were intravenously administered to 10 Balb/c nude mice bearing heterotopic subcutaneous human extrahepatic cholangiocarcinoma (TFK-1 and EGI-1) xenografts after which in vivo binding was assessed following contrast-enhanced destruction replenishment ultrasound application. Results Though not significantly associated with any examined clinicopathological variable, we found that higher CD105 expression was independently associated with poorer patient survival (median 12 vs 31 months; p = 0.002). In vitro studies revealed significant binding of CD105-microbubbles to SVR endothelial cells in comparison to isotype control (p = 0.01), as well as in vivo to TFK-1 (p = 0.02) and EGI-1 (p = 0.04) mouse xenograft vasculature. Conclusion Our results indicate that CD105 is a biomarker eminently suitable for cholangiocarcinoma targeting using functionalised microbubbles.

In silico modeling of izoniazid-steroid conjugates interactions with cytochromes P450 of mycobacteria and their bioconversion in vitro by the cells

Molecular docking of four hydrazones of isoniazid with steroids (dehydroepiandrosterone, pregnenolone, 16α,17α-epoxypregnenolone, cholestenone) — IDHEA, IPRE, IEP5, ICHN, to mycobacterial cytochromes P450 was performed. The in silico study has shown than these hydrazones can be effectively bound to CYP121, CYP124, CYP125, CYP126A1, CYP130, and CYP51 with binding energy ranged from -9 kcal/mol to -12 kcal/mol. Calculations also demonstrated enhancement of passive lipid bilayer permeability with respect to isoniazid. In vitro IDHEA, IPRE, IEPR were found to undergo bioconversion into their 3-keto-4-en derivatives. This suggests their ability to penetrate into M. tuberculosis H37Rv cells. The results of this study are important in the context of understanding of specificity of binding of synthetic steroid derivatives to mycobacterial CYPs and indicate the possibility of using the steroid compounds studied by us as new ligands for these enzymes.

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

Francisella novicidaCas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

SUMMARYGenome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely usedStreptococcus pyogenesCas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report thatFrancisella novicidaCas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.SIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 fromFrancisella novicida(FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.