Protein Substrates Research Articles

Substrate specificity and protein stability drive the divergence of plant-specific DNA methyltransferases.

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.

A proteolytic AAA+ machine poised to unfold protein substrates

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP’s ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX’s ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.

High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method.

Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.

ER Oxidoreductin 1-Like Activity of Cyclic Diselenides Drives Protein Disulfide Isomerase in an Electron Relay System.

Disulfide formation generally involves a two-electron oxidation reaction between cysteine residues. Additionally, disulfide formation is an essential post-translational modification for the structural maturation of proteins. This oxidative folding is precisely controlled by an electron relay network constructed by protein disulfide isomerase (PDI), with a CGHC sequence as the redox-active site, and its family enzymes. Creating reagents that mimic the functions of these enzymes facilitates folding during chemical protein synthesis. In this study, we aimed to imitate a biological electron relay system using cyclic diselenide compounds as surrogates for endoplasmic reticulum oxidoreductin 1 (Ero1), which is responsible for the re-oxidation of PDI. Oxidized PDI (PDIox) introduces disulfide bonds into substrate proteins, resulting in its conversion to reduced PDI (PDIred). The PDIred is then re-oxidized to PDIox by a coexisting cyclic diselenide compound, thereby restoring the function of PDI as a disulfide-forming agent. The produced diselenol state is readily oxidized to the original diselenide state with molecular oxygen, continuously sustaining the PDI catalytic cycle. This artificial electron relay system regulating enzymatic PDI function effectively promotes the oxidative folding of disulfide-containing proteins, such as insulin-a hypoglycemic formulation-by enhancing both yield and reaction velocity.

Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme.

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine's moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole's electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin's B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

Neddylation of protein, a new strategy of protein post-translational modification for targeted treatment of central nervous system diseases

Neddylation, a type of protein post-translational modification that links the ubiquitin-like protein NEDD8 to substrate proteins, can be involved in various significant cellular processes and generate multiple biological effects. Currently, the best-characterized substrates of neddylation are the Cullin protein family, which is the core subunit of the Cullin-RING E3 ubiquitin ligase complex and controls many important biological processes by promoting ubiquitination and subsequent degradation of various key regulatory proteins. The normal or abnormal process of protein neddylation in the central nervous system can lead to a series of occurrences of normal functions and the development of diseases, providing an attractive, reasonable, and effective targeted therapeutic strategy. Therefore, this study reviews the phenomenon of neddylation in the central nervous system and summarizes the corresponding substrates. Finally, we provide a detailed description of neddylation involved in CNS diseases and treatment methods that may be used to regulate neddylation for the treatment of related diseases.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

Potential application of room temperature synthesized MIL-100(Fe) in enhancing methane production in microbial electrolysis Cells-Anaerobic digestion treating Protein-Rich wastewater

Microbial electrolysis cell-anaerobic digestion (MEC-AD) is an emerging technology for methane production. However, low substrate degradation efficiency remains a challenge when processing protein substrates. This study developed a MIL-100(Fe) carbon cloth anode to enhance methane production and substrate degradation in MEC-AD. The effects of MIL-100(Fe) prepared under hydrothermal (H-MIL-100(Fe)) and room temperature conditions (R-MIL-100(Fe)) were compared. Results indicated that H-MIL-100(Fe) and R-MIL-100(Fe) increased cumulative methane production by 16.01% and 14.99%, respectively compared to normal cloth, each influencing methane production through distinct mechanisms. Electrochemical characterization showed that H-MIL-100(Fe) enhanced the electrochemical performance more significantly due to the enrichment of Geotalea, with the oxidation current improved by 7.39-fold (R-MIL-100(Fe) increased it by only 2.95-fold) to promote growth of Methanobacterium. Metagenomic analysis revealed that R-MIL-100(Fe) tended to metabolize amino acids into methane rather than support cellular life activities, indicating its practicality under limited substrate concentration. In summary, R-MIL-100(Fe) shows greater potential for application due to its mild synthesis conditions and advantages in treating complex substrates.

Degradome analysis to identify direct protein substrates of small-molecule degraders.

Targeted protein degradation (TPD) has emerged as a powerful strategy to selectively eliminate cellular proteins using small-molecule degraders, offering therapeutic promise for targeting proteins that are otherwise undruggable. However, a remaining challenge is to unambiguously identify primary TPD targets that are distinct from secondary downstream effects in the proteome. Here we introduce an approach for selective analysis of protein degradation by mass spectrometry (DegMS) at proteomic scale, which derives its specificity from the exclusion of confounding effects of altered transcription and translation induced by target depletion. We show that the approach efficiently operates at the timescale of TPD (hours) and we demonstrate its utility by analyzing the cyclin K degraders dCeMM2 and dCeMM4, which induce widespread transcriptional downregulation, and the GSPT1 degrader CC-885, an inhibitor of protein translation. Additionally, we apply DegMS to characterize a previously uncharacterized degrader, and identify the zinc-finger protein FIZ1 as a degraded target.

Allosteric modulation of the Lon protease via ssDNA binding and local charge changes.

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduce ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements, we find this change in activity correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.

Bioorthogonal Monomycolate of Trehalose Disclosed the O-Mycoloylation of Mycoloyltransferases and Other Cell Envelope Proteins in C. glutamicum.

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Many questions about the specificity of protein O-mycoloylation remain crucial for understanding its evolutionary significance in Mycobacteriales and its role in cell physiology. We have developed the first bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct, unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ΔmytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

Role and therapeutic potential of E3s in the tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a high-incidence, poor-prognosis malignancy worldwide, requiring new strategies for treatment. Ubiquitination, especially ubiquitination through E3 ubiquitin ligases, plays an indispensable role in the development and progression of HCC. E3 ubiquitin ligases are crucial enzymes in ubiquitination, controlling the degradation of specific substrate proteins and influencing various cellular functions, such as tumor cell proliferation, apoptosis, migration, and immune evasion. In this review, we systematically summarize the mechanisms of E3 ubiquitin ligases in HCC, with a focus on the significance of RING, HECT, and RBR types in HCC progression. The review also looks at the potential for targeting E3 ligases to modulate the tumor microenvironment (TME) and increase immunotherapy efficacy. Future studies will optimize HCC treatment by formulating specific inhibitors or approaches that will be based on gene therapy targeting E3 ligases in order to overcome resistance issues with present treatments and create optimism in the journey of treatment for HCC patients.

Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their β-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the β subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification.

High-level secretory expression and characterization of an acid protease in Komagataella phaffii and its application in soybean meal protein degradation

Acid proteases play a crucial role in the industrial enzyme market, but low yield limits their widespread application. In this study, we focused on enhancing the secretory expression level of an acid protease (AopepA) from Aspergillus oryzae in Komagataella phaffii through stepwise genetic modification strategies. These included the co-expression of endoplasmic reticulum secretion-associated factors, overexpression of eukaryotic translation initiation factors, knockout of the β-1,3-glucanosyltransferase gene, disruption of the hypoxic heme-dependent repressor gene, and co-expression of the hemoglobin gene. After these modifications, protease activity increased by 4.2-fold, reaching 536.6 U/mL in a shaking flask. The engineered strain produced protease activity of up to 17,392.0 U/mL with a protein concentration of 44.6 g/L in a 5 L fermenter, representing the highest secretory expression level of acid proteases in K. phaffii ever reported. The optimal conditions of AopepA were pH 3.0 and 50 °C. AopepA demonstrated broad hydrolysis activity towards various protein substrates. It efficiently degraded soybean meal proteins into low molecular weight (Mw < 1 kDa, accounting for 82 %) oligopeptides to enhance protein utilization. This study provides valuable insights into improving the secretory expression of acid proteases in K. phaffii and identifies a suitable acid protease for enhancing soybean meal protein utilization.

The Investigation of Hsp90C-Terminal Inhibitors Containing Amide Bioisosteres.

Heat Shock Protein 90 (Hsp90) is responsible for the proper folding and maturation of ~400 client protein substrates, many of which are directly associated with the ten hallmarks of cancer. Hsp90 is a great target for cancer therapy including melanoma, since Hsp90 inhibition can disrupt multiple oncogenic pathways simultaneously. In this study, we report the synthesis and anti-proliferative activity manifested by a series of Hsp90 C-terminal inhibitors against mutant BRAF and wild-type BRAF melanoma cells. Furthermore, we explored structure-activity relationships (SAR) for the amide moiety of 6 (B1), a novel Hsp90C-terminal inhibitor via introduction of amide bioisosteres. Compound 6 displayed an IC50 of 1.01 μM, 0.782 μM, 0.607 μM and 1.413 μM against SKMel173, SKMel103, SKMel19 and A375 cells, respectively.

Involvement of E3 Ubiquitin Ligases in Viral Infections of the Human Host.

Viral infections are one of the principal causes of global primary health crises, with increased rate of infection and mortality demonstrated by the newer progeny of viruses. Viral invasion of the host involves utilization of various cellular machinery. Ubiquitination is one of a few central regulatory systems used by viruses for establishment of the infections in the host. Members of the ubiquitination system are involved in carrying out proteasomal degradation or functional modification of proteins in numerous cellular processes. E3 ubiquitin ligases play a major role in this system through recognition and recruitment of protein substrates and catalyzing the transfer of ubiquitin to these substrates. The versatility of ubiquitin ligases frequently makes them useful tools for the viruses, for either utilizing or degrading other cellular machineries, for carrying out their multiplication or inactivating the defensive strategies of the host. Therefore, these ligases are important targets for aiming at major pathways causing viral protein degradation or functional modification of the infection process. In this review, we have discussed the role and mechanism of different types of ubiquitin ligases in the context of infections of mainly human viruses, highlighting the viral proteins directly interacting with the ligases. Knowledge about these direct interactions is central in understanding the ubiquitin-dependent processes. This comprehensive account may also be beneficial for pharmaceutical exploration of E3 ligase-based broad-spectrum antiviral treatment.

Protein kinases as therapeutic targets for Alzheimer’s disease: a brief review

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder, with an unknown etiology and a multifactorial pathophysiology characterized by protein misfolding, neuroinflammation, and neuronal loss. There are three well-discussed main hypotheses for the pathophysiology of AD, which are related to i) the accumulation of amyloid β (Aβ) protein aggregates in the extracellular space, ii) deposition of hyperphosphorylated tau fragments as neurofibrillary tangles, and iii) dysregulation of hemostasis of some neurotransmitters involved in the disease, such as acetylcholine (ACh) and glutamate. The association of all these factors is responsible for installing oxidative stress and neuroinflammation, which contribute to progressive neuronal death in specific brain regions. More recently, other remarkable pathological characteristics have been described, involving changes in all levels of cellular components, especially in the action and function of protein kinases. These enzymes are crucial for cellular regulation since they play a pivotal role in the phosphorylation of protein substrates by transferring a phosphate group from the ATP molecule to threonine, serine, or tyrosine residues. In more recent studies, some kinases have been especially reported by their role in inflammatory and oxidative processes associated to AD, such as cAMP-dependent protein kinase A (PKA), cyclin-dependent protein kinase 5 (CDK5), glycogen synthase kinase 3β (GSK-3β), and the microtubule affinity regulatory kinases (MARKs). Under homeostatic conditions, protein kinases act as cellular signals, directing physiological responses, but in AD pathogenesis, these enzymes have an exacerbated activity in the brain, justifying the need for a better comprehension of their function and role, and how new kinase inhibitors could lead to innovative drugs. In this context, this brief review aimed to compile the literature data related to the most recent efforts and strategies in Medicinal Chemistry in the discovery of new kinase inhibitors, opening new ways to AD therapeutics.

Hydrolysates of mare's milk proteins. Immunochemical and physico-chemical characteristics

In recent years, the attention of specialists has been attracted by a significant increase in allergic diseases, especially among children. Enzymatic hydrolysis with the participation of peptidases is considered the most effective measure to reduce the allergenicity of milk proteins. The study of the features of protein proteolysis by various enzymes, aimed at establishing optimal process parameters, is the subject of research in the production of hydrolysates with specified properties: a certain peptide and amino acid profile, reduced antigenic properties. At the same time, for a correct preclinical assessment of the safety and potential effectiveness of specialized food products, it is not enough only to determine the molecular weight distribution of peptide fractions in the composition. A necessary additional and informative indicator of reducing the potential allergenicity of the hydrolysate is the degree of preservation of the antigenic properties of the initial protein (residual antigenicity), determined by immunochemical methods. The preparation and subsequent comprehensive assessment of enzymatic hydrolysates of mare's milk proteins is of considerable interest, since, according to the results of clinical studies, it was suggested that upon allergy to cow's milk proteins mare's milk can be considered hypoallergenic. The purpose of this work was to obtain enzymatic hydrolysates of mare's milk proteins using domestic enzymes and their further physico-chemical and immunochemical characteristics. The object of the study was mare's milk protein obtained from skimmed mare's milk powder as a protein substrate. Two samples of enzymes from various manufacturers of the Russian Federation were used in the work. The results of the conducted studies show that the peptide profiles of hydrolysates vary greatly depending on the enzyme used, however, the multiplicity of antigenicity reduction practically does not depend on the nature of the enzyme, and does not depend on an increase in the enzyme/substrate ratio. The peptide profile and the multiplicity of reducing the antigenicity of hydrolysates of mare's milk proteins are influenced by ultrafiltration treatment of the obtained hydrolysates, which allows reducing the content of antigenic structures in the final product by several orders of magnitude.

Current trends to design antimalarial drugs targeting N-myristoyltransferase.

Malaria is a disease caused by Plasmodium spp., of which Plasmodium falciparum and Plasmodium vivax are the most prevalent. Unfortunately, traditional and some current treatment regimens face growing protozoan resistance. Thus, searching for and exploring new drugs and targets is necessary. One of these is N-myristoyltransferase (NMT). This enzyme is responsible for the myristoylation of several protein substrates in eukaryotic cells, including Plasmodium spp., thus enabling the assembly of protein complexes and stabilization of protein-membrane interactions. Given the importance of this target in developing new antiparasitic drugs, this review aims to explore the recent advances in the design of antimalarial drugs to target Plasmodium NMT.

PGC-1α regulation by FBXW7 through a novel mechanism linking chaperone-mediated autophagy and the ubiquitin-proteasome system.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and antioxidative defenses, and it may play a critical role in Parkinson's disease (PD). F-box/WD repeat domain-containing protein (FBXW7), an E3 protein ligase, promotes the degradation of substrate proteins through the ubiquitin-proteasome system (UPS) and leads to the clearance of PGC-1α. Here, we elucidate a novel post-translational mechanism for regulating PGC-1α levels in neurons. We show that enhancing chaperone-mediated autophagy (CMA) activity promotes the CMA-mediated degradation of FBXW7 and consequently increases PGC-1α. We confirm the relevance of this pathway in vivo by showing decreased FBXW7 and increased PGC-1α as a result of boosting CMA selectively in dopaminergic (DA) neurons by overexpressing lysosomal-associated membrane protein 2A (LAMP2A) in TH-Cre-LAMP2-loxp conditional mice. We further demonstrate that these mice are protected against MPTP-induced oxidative stress and neurodegeneration. These results highlight a novel regulatory pathway for PGC-1α in DA neurons and suggest targeted increasing of CMA or decreasing FBXW7 in DA neurons as potential neuroprotective strategies in PD.