Substrate Sequence Research Articles

DNA-Encoded Noncanonical Substrate Library for Protease Profiling.

Profiling the substrate sequence preferences of proteases is important for understanding both biological functions as well as for designing protease inhibitors. Several methods are available for profiling the sequence specificity of proteases. However, there is currently no rapid and high-throughput method to profile specificity of proteases for noncanonical substrates. In this study, we described a strategy to use a DNA-encoded noncanonical substrate library to identify the protease substrates composed of both canonical and noncanonical amino acids. This approach uses a DNA-encoded peptide library and introduces a biotin molecule at the N-terminus to immobilize the library on a solid support. Upon protease hydrolysis, the released DNA tag of the substrate peptides can be sequenced to identify the substrate structures. Using this approach, we profiled trypsin and fibroblast activation protein α and discovered noncanonical substrates that were more efficiently cleaved than the commonly used substrates. The identified substrates of FAP were further used to design corresponding covalent inhibitors containing non-canonical sequences with high potency for the target protease. Overall, our approach can aid in the development of new protease substrates and inhibitors.

Closing in on human methylation-the versatile family of seven-β-strand (METTL) methyltransferases.

Methylation is a common biochemical reaction, and a number of methyltransferase (MTase) enzymes mediate the various methylation events occurring in living cells. Almost all MTases use the methyl donor S-adenosylmethionine (AdoMet), and, in humans, the largest group of AdoMet-dependent MTases are the so-called seven-β-strand (7BS) MTases. Collectively, the 7BS MTases target a wide range of biomolecules, i.e. nucleic acids and proteins, as well as severalsmall metabolites and signaling molecules. They play essential roles in key processes such as gene regulation, protein synthesis and metabolism, as well as neurotransmitter synthesis and clearance. A decade ago, roughlyhalf of the human 7BS MTases had been characterized experimentally, whereas the remaining ones merely represented hypothetical enzymes predicted from bioinformatics analysis, many of which were denoted METTLs (METhylTransferase-Like). Since then, considerable progress has been made, and the function of>80% of the human 7BS MTases has been uncovered. In this review, I provide an overview of the (estimated) 120 human 7BS MTases, grouping them according to substrate specificities and sequence similarity. I also elaborate on the challenges faced when studying these enzymes and describe recent major advances in the field.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

How the double-ring ClpAP protease motor grips the substrate to unfold and degrade stable proteins

Loops in the axial channels of ClpAP and other AAA+ proteases bind a short peptide degron connected by a linker to the N- or C-terminal residue of a native protein to initiate degradation. ATP hydrolysis then powers pore-loop movements that translocate these segments through the channel until a native domain is pulled against the narrow channel entrance, creating an unfolding force. Substrate unfolding is thought to depend on strong contacts between pore loops and a subset of amino acids in the unstructured sequence directly preceding the folded domain. Here, we identify such contact sequences that promote grip for ClpAP and use ClpA structures to place these sequences within ClpA's two AAA+ rings. The positions and chemical nature of certain residues within an unstructured segment that are positioned to interact with the D2 ring have major positive effects on substrate unfolding, whereas segments located within the D1 ring have little consequence. Within the D2-bound segment, two short elements are critical for accelerating degradation; one is at the 'top' of D2 and consists of at least two properly positioned non-slippery residues. In contrast, the second D2 element, which can be as short as one residue, is positioned to contact pore loops near the 'bottom' of this ring. Comparison with similar studies for ClpXP reveals that positioning a well-gripped substrate sequence within the major unfoldase motor is more important than its proximity to the folded domain and that charged, polar, and hydrophobic residues all contribute favorable contacts to substrate grip.

Design of a triple-functional polyA-probe: Toward a facile electrochemical aptasensor for vanillin detection

This study successfully developed an electrochemical aptasensor based on a triple-functional biotin-modified poly-adenine (polyA) probe (Bio-TPP) for one-step ultrasensitive detection of vanillin (VAN). The Bio-TPP was self-assembled onto a gold electrode (AuE) via its polyA sequence, followed by conjugation with PtPd@Ni-Co layered double hydroxides labeled with streptavidin and thionine (SA/Thi/PtPd@Ni-Co LDH), which generated electrochemical signals. Importantly, in the presence of VAN, the specific substrate sequence of a Mg²⁺-dependent DNAzyme in the Bio-TPP hybridized with the output DNAzyme strand generated by the specific recognition between the aptamer (Apt) and VAN. And, with the help of Mg²⁺, the DNAzyme amplification reaction was triggered. This led to a significant reduction in the electrochemical signals, enabling highly sensitive detection of VAN. Benefiting from the precise and flexible design of the Bio-TPP, this aptasensor offered a new detection method, exhibiting excellent performance with a wide linear range of 1×10−6-10 μM and a detection limit of 0.30 pM. Additionally, this assay possessed good selectivity, reproducibility, and stability, which held great potential in bioanalysis.

Assay Development and Validation for Innovative Antiviral Development Targeting the N-Terminal Autoprocessing of SARS-CoV-2 Main Protease Precursors.

The SARS-CoV-2 main protease (Mpro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature Mpro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 Mpro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released Mpro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released Mpro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 Mpro precursor autoprocessing or the released mature Mpro through different mechanisms of action.

Insights into the DNA and RNA Interactions of Human Topoisomerase III Beta Using Molecular Dynamics Simulations.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.

Discovery of NSD2 non-histone substrates and design of a super-substrate

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.

Guiding Transient Peptide Assemblies with Structural Elements Embedded in Abiotic Phosphate Fuels

AbstractDespite great progress in the construction of non‐equilibrium systems, most approaches do not consider the structure of the fuel as a critical element to control the processes. Herein, we show that the amino acid side chains (A, F, Nal) in the structure of abiotic phosphates can direct assembly and reactivity during transient structure formation. The fuels bind covalently to substrates and subsequently influence the structures in the assembly process. We focus on the ways in which the phosphate esters guide structure formation and how structures and reactivity cross regulate when constructing assemblies. Through the chemical functionalization of energy‐rich aminoacyl phosphate esters, we are able to control the yield of esters and thioesters upon adding dipeptides containing tyrosine or cysteine residues. The structural elements around the phosphate esters guide the lifetime of the structures formed and their supramolecular assemblies. These properties can be further influenced by the peptide sequence of substrates, incorporating anionic, aliphatic and aromatic residues. Furthermore, we illustrate that oligomerization of esters can be initiated from a single aminoacyl phosphate ester incorporating a tyrosine residue (Y). These findings suggest that activated amino acids with varying reactivity and energy contents can pave the way for designing and fabricating structured fuels.

Guiding Transient Peptide Assemblies with Structural Elements Embedded in Abiotic Phosphate Fuels.

Despite great progress in the construction of non-equilibrium systems, most approaches do not consider the structure of the fuel as a critical element to control the processes. Herein, we show that the amino acid side chains (A, F, Nal) in the structure of abiotic phosphates can direct assembly and reactivity during transient structure formation. The fuels bind covalently to substrates and subsequently influence the structures in the assembly process. We focus on the ways in which the phosphate esters guide structure formation and how structures and reactivity cross regulate when constructing assemblies. Through the chemical functionalization of energy-rich aminoacyl phosphate esters, we are able to control the yield of esters and thioesters upon adding dipeptides containing tyrosine or cysteine residues. The structural elements around the phosphate esters guide the lifetime of the structures formed and their supramolecular assemblies. These properties can be further influenced by the peptide sequence of substrates, incorporating anionic, aliphatic and aromatic residues. Furthermore, we illustrate that oligomerization of esters can be initiated from a single aminoacyl phosphate ester incorporating a tyrosine residue (Y). These findings suggest that activated amino acids with varying reactivity and energy contents can pave the way for designing and fabricating structured fuels.

A one-process production of completely biotinylated proteins in a T7 expression system.

Streptavidin is a tetrameric protein with high specificity and affinity for biotin. The interaction between avidin and biotin has become a valuable tool in nanotechnology. In recent years, the site-specific biotin modification of proteins using biotin ligases, such as BirA, has attracted attention. This study established an in vivo method for achieving the complete biotinylation of target proteins using a single plasmid co-expressing BirA and its target proteins. Specifically, a biotin-modified protein was produced in Escherichia coli strain BL21(DE3) using a single plasmid containing genes encoding both BirA and a protein fused to BirA's substrate sequence, Avitag. This approach simplifies the production of biotinylated proteins in E. coli and allows the creation of various biotinylated protein types through gene replacement. Furthermore, the biotin modification rate of the obtained target protein could be evaluated using Native-PAGE without performing complicated isolation operations of biotinylated proteins. In Native-PAGE, biotin-modified proteins and unmodified proteins were confirmed as clearly different bands, and it was possible to easily derive the modification rate from the respective band intensities.

The intrinsic substrate specificity of the human tyrosine kinome

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1–3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4–7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt.

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

Substrate Selection Criteria in Regulated Intramembrane Proteolysis.

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aβ peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

Mechanism of Mutation-Induced Effects on the Catalytic Function of TEV Protease: A Molecular Dynamics Study.

Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp's ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.

A resource database for protein kinase substrate sequence-preference motifs based on large-scale mass spectrometry data

BackgroundProtein phosphorylation is one of the most prevalent posttranslational modifications involved in molecular control of cellular processes, and is mediated by over 520 protein kinases in humans and other mammals. Identification of the protein kinases responsible for phosphorylation events is key to understanding signaling pathways. Unbiased phosphoproteomics experiments have generated a wealth of data that can be used to identify protein kinase targets and their preferred substrate sequences.MethodsThis study utilized prior data from mass spectrometry-based studies identifying sites of protein phosphorylation after in vitro incubation of protein mixtures with recombinant protein kinases. PTM-Logo software was used with these data to generate position-dependent Shannon information matrices and sequence motif ‘logos’. Webpages were constructed for facile access to logos for each kinase and a new stand-alone application was written in Python that uses the position-dependent Shannon information matrices to identify kinases most likely to phosphorylate a particular phosphorylation site.ResultsA database of kinase substrate target preference logos allows browsing, searching, or downloading target motif data for each protein kinase (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/). These logos were combined with phylogenetic analysis of protein kinase catalytic sequences to reveal substrate preference patterns specific to particular groups of kinases (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinaseTree.html). A stand-alone program, KinasePredictor, is provided (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinasePredictor.html). It takes as input, amino-acid sequences surrounding a given phosphorylation site and generates a ranked list of protein kinases most likely to phosphorylate that site.ConclusionsThis study provides three new resources for protein kinase characterization. It provides a tool for prediction of kinase-substrate interactions, which in combination with other types of data (co-localization, etc.), can predict which kinases are likely responsible for a given phosphorylation event in a given tissue.-q3N7pjxaL8wVBVRsJ9wBTVideo

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

A novel fluorescent biosensor for detection of Pb2+ based on Pb2+-specific DNAzyme and carbon dots prepared from watermelon rind

Abstract A novel fluorescent biosensor was constructed to detect Pb2+. Multifunctional magnetic beads modified with substrate sequence and Pb2+-specific DNAzyme were employed as recognition probes. The fluorescence of carbon dots synthesized from watermelon rind (W-CDs) was quenched by hemin. Hemin/W-CDs were employed as fluorescent signals. The presence of Pb2+ could target-trigger the biosensor, and generated G-quadruplex which could restore the fluorescence of hemin/W-CDs. The fluorescence change of the biosensor depended on Pb2+ concentration from 1 to 20 nM.

DNAzyme-Catalyzed Site-Specific N-Acylation of DNA Oligonucleotide Nucleobases.

We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6-tetrafluorophenyl ester (TFPE) of a 5'-carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N-acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site-specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site-specific N-acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N-acylation, toward a universal approach for site-specific oligonucleotide modification.

DNAzyme‐Catalyzed Site‐Specific N‐Acylation of DNA Oligonucleotide Nucleobases

AbstractWe used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6‐tetrafluorophenyl ester (TFPE) of a 5′‐carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N‐acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site‐specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site‐specific N‐acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N‐acylation, toward a universal approach for site‐specific oligonucleotide modification.