Increased Protein Stability Research Articles

Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

Simple SummaryResistance to tyrosine kinase inhibitors (TKIs) that target common non-small-cell lung cancer mutations within the epidermal growth factor receptor (EGFR) is a primary clinical issue. The aim of our study was to determine whether the protein cell division cycle-associated protein 3 (CDCA3) might be a biomarker for TKI response in EGFR mutant lung cancer. Our previous work has demonstrated that CDCA3 is a marker of chemotherapy sensitivity in lung cancer. We provide evidence that CDCA3 levels are increased in EGFR mutant lung cancer and these levels are associated with sensitivity to TKIs. In addition, increasing the levels of CDCA3 enhances TKI sensitivity in models of TKI-resistant EGFR mutant lung cancer. Our findings propose that strategies to upregulate CDCA3 levels might improve TKI response in EGFR mutant lung cancer.Tyrosine kinase inhibitors (TKIs) are the first-line therapy for non-small-cell lung cancers (NSCLC) that harbour sensitising mutations within the epidermal growth factor receptor (EGFR). However, resistance remains a key issue, with tumour relapse likely to occur. We have previously identified that cell division cycle-associated protein 3 (CDCA3) is elevated in adenocarcinoma (LUAD) and correlates with sensitivity to platinum-based chemotherapy. Herein, we explored whether CDCA3 levels were associated with EGFR mutant LUAD and TKI response. We demonstrate that in a small-cohort tissue microarray and in vitro LUAD cell line panel, CDCA3 protein levels are elevated in EGFR mutant NSCLC as a result of increased protein stability downstream of receptor tyrosine kinase signalling. Here, CDCA3 protein levels correlated with TKI potency, whereby CDCA3high EGFR mutant NSCLC cells were most sensitive. Consistently, ectopic overexpression or inhibition of casein kinase 2 using CX-4945, which pharmacologically prevents CDCA3 degradation, upregulated CDCA3 levels and the response of T790M(+) H1975 cells and two models of acquired resistance to TKIs. Accordingly, it is possible that strategies to upregulate CDCA3 levels, particularly in CDCA3low tumours or upon the emergence of therapy resistance, might improve the response to EGFR TKIs and benefit patients.

ERRα contributes to HDAC6-induced chemoresistance of osteosarcoma cells.

Chemotherapy resistance is an important problem for clinical therapy of osteosarcoma (OS). The potential effects of histone deacetylases (HDACs) on OS chemoresistance are studied. The expression of HDACs in OS cells resistance to doxorubicin (Dox) and cisplatin (CDDP) is checked. Among 11 members of HDACs, levels of HDAC6 are significantly upregulated in OS cells resistance to Dox and CDDP. Inhibition of HDAC6 via its specific inhibitor ACY1215 restores chemosensitivity of OS-resistant cells. Further, HDAC6 directly binds with estrogen-related receptors alpha (ERRα) to regulate its acetylation and protein stability. Inhibition of ERRα further strengthens ACY1215-increased chemosensitivity of OS-resistant cells. Mechanistically, K129 acetylation is the key residue for HDAC6-regulated protein levels of ERRα. Collectively, we find that ERRα contributes to HDAC6-induced chemoresistance of OS cells. Inhibition of HDAC6/ERRα axis might be a potential approach to overcome chemoresistance and improve therapy efficiency for OS treatment. 1. HDAC6 was significantly upregulated in Dox and CDDP resistant OS cells; 2. Inhibition of HDAC6 can restore chemosensitivity of OS cells; 3. HDAC6 binds with ERRα at K129 to decrease its acetylation and increase protein stability; 4. ERRα contributes to HDAC6-induced chemoresistance of OS cells.

SARS-CoV-2 Variants Are Selecting for Spike Protein Mutations That Increase Protein Stability.

The emergence of variants of SARS-CoV-2 with mutations in their spike protein are a major cause for concern for the efficacy of vaccines and control of the pandemic. We show that mutations in the spike protein of SARS-CoV-2 are selecting for amino acid changes that result in a more thermodynamically stable protein than expected from background. We suggest that the computationally efficient analysis of mutational stability may aid in early screening of variants.

Coilin enhances phosphorylation and stability of DGCR8 and promotes miRNA biogenesis.

MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is composed of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of coimmunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.

Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

RNA-binding proteins play crucial roles in various cellular functions and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15), as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3. Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, whereas underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV–S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA‐binding proteins

Over 50% of RNA-binding protein regions are disordered and characterized by repetitive sequences. We found that the most dominant repetitive sequences are electronegative clusters (ENCs) containing acidic residues and/or phosphorylation sites. Despite their abundance, ENCs’ functions are largely unknown. We elucidated the regulatory functions of ENCs using ribosomal biogenesis factor Nop15 as a model. Nop15 has an ENC and an RNA-recognition motif that binds to ITS2 RNA. We found that Nop15's ENC increases protein stability, and inhibits nonspecific RNA binding, but minimally affects specific binding. The ENC performs these regulatory functions through intramolecular interactions. We proposed that ENCs are selected by evolution for suppressing nonspecific binding. Consistent with this proposal, ENCs are also overrepresented in DNA-binding proteins, but underrepresented in halophiles in which nonspecific binding is less problematic. Using the high-throughput RNA Bind-n-Seq method, we investigated how an engineered ENC modulates the sequence specificity of RNA binding. We found that the engineered ENC increases RNA-binding specificity by downplaying the role of electrostatic interactions. In summary, our work revealed a general regulatory mechanism of ENCs. Considering that the regulatory function of ENCs depends on their length, ENCs can be conveniently used for design of more stable and specific RNA-binding proteins.

The Diarylheptanoid Curcumin Induces MYC Inhibition and Cross-Links This Oncoprotein to the Coactivator TRRAP.

The c-Myc protein (MYC) is a transcription factor with strong oncogenic potential controlling fundamental cellular processes. In most human tumors, MYC is overexpressed by enhanced transcriptional activation, gene amplification, chromosomal rearrangements, or increased protein stabilization. To pharmacologically suppress oncogenic MYC functions, multiple approaches have been applied either to inhibit transcriptional activation of the endogenous MYC gene, or to interfere with biochemical functions of aberrantly activated MYC. Other critical points of attack are targeted protein modification, or destabilization leading to a non-functional MYC oncoprotein. It has been claimed that the natural compound curcumin representing the principal curcumoid of turmeric (Curcuma longa) has anticancer properties although its specificity, efficacy, and the underlying molecular mechanisms have been controversially discussed. Here, we have tested curcumin’s effect on MYC-dependent cell transformation and transcriptional activation, and found that this natural compound interferes with both of these MYC activities. Furthermore, in curcumin-treated cells, the endogenous 60-kDa MYC protein is covalently and specifically cross-linked to one of its transcriptional interaction partners, namely the 434-kDa transformation/transcription domain associated protein (TRRAP). Thereby, endogenous MYC levels are strongly reduced and cells stop to proliferate. TRRAP is a multidomain adaptor protein of the phosphoinositide 3-kinase-related kinases (PIKK) family and represents an important component of many histone acetyltransferase (HAT) complexes. TRRAP is important to mediate transcriptional activation executed by the MYC oncoprotein, but on the other hand TRRAP also negatively regulates protein stability of the tumor suppressor p53 (TP53). Curcumin-mediated covalent binding of MYC to TRRAP reduces the protein amounts of both interaction partners but does not downregulate TP53, so that the growth-arresting effect of wild type TP53 could prevail. Our results elucidate a molecular mechanism of curcumin action that specifically and irreversibly targets two crucial multifunctional cellular players. With regard to their broad impact in cancer, our findings contribute to explain the pleiotropic functions of curcumin, and suggest that this natural spice, or more bioavailable derivatives thereof, may constitute useful adjuvants in the therapy of MYC-dependent and TRRAP-associated human tumors.

Molecular Dynamics-Guided Design of a Functional Protein-ATRP Conjugate That Eliminates Protein-Protein Interactions.

Even the most advanced protein-polymer conjugate therapeutics do not eliminate antibody-protein and receptor-protein recognition. Next-generation bioconjugate drugs will need to replace stochastic selection with rational design to select desirable levels of protein-protein interaction while retaining function. The "Holy Grail" for rational design would be to generate functional enzymes that are fully catalytic with small molecule substrates while eliminating interaction between the protein surface and larger molecules. Using chymotrypsin, an important enzyme that is used to treat pancreatic insufficiency, we have designed a series of molecular chimeras with varied grafting densities and shapes. Guided by molecular dynamic simulations and next-generation molecular chimera characterization with asymmetric flow field-flow fractionation chromatography, we grew linear, branched, and comb-shaped architectures from the surface of the protein by atom-transfer radical polymerization. Comb-shaped polymers, grafted from the surface of chymotrypsin, completely prevented enzyme inhibition with protein inhibitors without sacrificing the ability of the enzyme to catalyze the hydrolysis of a peptide substrate. Asymmetric flow field-flow fractionation coupled with multiangle laser light scattering including dynamic light scattering showed that nanoarmor designed with comb-shaped polymers was particularly compact and spherical. The polymer structure significantly increased protein stability and reduced protein-protein interactions. Atomistic molecular dynamic simulations predicted that a dense nanoarmor with long-armed comb-shaped polymer would act as an almost perfect molecular sieve to filter large ligands from substrates. Surprisingly, a conjugate that was composed of 99% polymer was needed before the elimination of protein-protein interactions.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

High glucose activates ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic foot ulcers

Increased matrix metalloproteinase 9 (MMP9) expression is involved in delayed wound healing in diabetic foot ulcers. We created skin wounds in normal SD rats and STZ-induced diabetic SD rats, then we found protein levels of activator protein-1 (AP1), a crucial transcription factor to increase MMP9 transcription, as well as MMP9 was up-regulated in epithelium of diabetic skin tissues. Then, we evaluated the mRNA and protein stability of AP1 subunits C-FOS/C-Jun in HaCaT cells after high glucose treatment. Results showed that high glucose could increase protein stability of C-FOS and C-Jun. Additionally, high glucose also activated extracellular signaling-related kinase 1/2 (ERK1/2). ERK1/2 inhibitor could rescue phosphorylation of C-FOS and C-Jun, increased protein stability of C-Jun, and increased MMP9 expressions. Thus, our study demonstrated that high glucose could activate ERK1/2 to stabilize AP1 and increase MMP9 expression in diabetic skin and HaCaT cells.

AtaT Improves the Stability of Pore-Forming Protein EspB by Acetylating Lysine 206 to Enhance Strain Virulence.

A novel type II toxin of toxin–antitoxin systems (TAs), Gcn5-related N-acetyltransferase (GNAT) family, was reported recently. GNAT toxins are mainly present in pathogenic species, but studies of their involvement in pathogenicity are rare. This study discovered that the GANT toxin AtaT in enterohemorrhagic Escherichia coli (EHEC) can significantly enhance strain pathogenicity. First, we detected the virulence of ΔataT and ΔataR in cell and animal models. In the absence of ataT, strains showed a lower adhesion number, and host cells presented weaker attaching and effacing lesions, inflammatory response, and pathological injury. Next, we screened the acetylation substrate of AtaT to understand the underlying mechanism. Results showed that E. coli pore-forming protein EspB, which acts as a translocon in type III secretion system (T3SS) in strains, can be acetylated specifically by AtaT. The acetylation of K206 in EspB increases protein stability and maintains the efficiency of effectors translocating into host cells to cause close adhesion and tissue damage.

Clinical utility of TGFB1 and its receptors (TGFBR1 and TGFBR2) in thyroid nodules: evaluation based on single nucleotide polymorphisms and mRNA analysis.

Abnormalities involving the TGFB1 gene and its receptors are common in several types of cancer and often related to tumor progression. We investigated the role of single nucleotide polymorphisms (SNP) in the susceptibility to cancer, their impact on its features, as well as the role of mRNA expression of these genes in thyroid malignancy. We genotyped TGFB1, TGFBR1, and TGFBR2 SNPs in 157 papillary thyroid cancer (PTC) patients and 200 healthy controls. Further, we investigated RNA samples of 47 PTC and 80 benign nodules, searching for differential mRNA expression. SNPs rs1800472 and rs1800469 were associated with characteristics of PTC aggressiveness. Effect predictor software analysis of nonsynonymous SNP rs1800472 indicated increasing protein stability and post-translational changes. TGFB1 mRNA expression was upregulated in PTC and downregulated in benign samples, differentiating malignant from benign nodules (p<0.0001); PTC from goiter (p<0.0001); and PTC from FA (p<0.0001). TGFBR1 mRNA expression was upregulated in goiter and PTC, but downregulated in FA, distinguishing PTC from goiter (p=0.0049); PTC from FA (p<0.0001); and goiter from FA (p=0.0267). On the other hand, TGFBR2 was downregulated in all histological types analyzed and was not able to differentiate thyroid nodules. TGFB1 polymorphism rs1800472 may confer greater activity to TGF-β1 in the tumor microenvironment, favoring PTC aggressiveness. Evaluation of TGFB1 and TGFBR1 mRNA levels may be useful to identify malignancy in thyroid nodules.

Comparative Genomics and Integrated Network Approach Unveiled Undirected Phylogeny Patterns, Co-mutational Hot Spots, Functional Cross Talk, and Regulatory Interactions in SARS-CoV-2.

ABSTRACTThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has resulted in 92 million cases in a span of 1 year. The study focuses on understanding population-specific variations attributing its high rate of infections in specific geographical regions particularly in the United States. Rigorous phylogenomic network analysis of complete SARS-CoV-2 genomes (245) inferred five central clades named a (ancestral), b, c, d, and e (subtypes e1 and e2). Clade d and subclade e2 were found exclusively comprised of U.S. strains. Clades were distinguished by 10 co-mutational combinations in Nsp3, ORF8, Nsp13, S, Nsp12, Nsp2, and Nsp6. Our analysis revealed that only 67.46% of single nucleotide polymorphism (SNP) mutations were at the amino acid level. T1103P mutation in Nsp3 was predicted to increase protein stability in 238 strains except for 6 strains which were marked as ancestral type, whereas co-mutation (P409L and Y446C) in Nsp13 were found in 64 genomes from the United States highlighting its 100% co-occurrence. Docking highlighted mutation (D614G) caused reduction in binding of spike proteins with angiotensin-converting enzyme 2 (ACE2), but it also showed better interaction with the TMPRSS2 receptor contributing to high transmissibility among U.S. strains. We also found host proteins, MYO5A, MYO5B, and MYO5C, that had maximum interaction with viral proteins (nucleocapsid [N], spike [S], and membrane [M] proteins). Thus, blocking the internalization pathway by inhibiting MYO5 proteins which could be an effective target for coronavirus disease 2019 (COVID-19) treatment. The functional annotations of the host-pathogen interaction (HPI) network were found to be closely associated with hypoxia and thrombotic conditions, confirming the vulnerability and severity of infection. We also screened CpG islands in Nsp1 and N conferring the ability of SARS-CoV-2 to enter and trigger zinc antiviral protein (ZAP) activity inside the host cell.IMPORTANCE In the current study, we presented a global view of mutational pattern observed in SARS-CoV-2 virus transmission. This provided a who-infect-whom geographical model since the early pandemic. This is hitherto the most comprehensive comparative genomics analysis of full-length genomes for co-mutations at different geographical regions especially in U.S. strains. Compositional structural biology results suggested that mutations have a balance of opposing forces affecting pathogenicity suggesting that only a few mutations are effective at the translation level. Novel HPI analysis and CpG predictions elucidate the proof of concept of hypoxia and thrombotic conditions in several patients. Thus, the current study focuses the understanding of population-specific variations attributing a high rate of SARS-CoV-2 infections in specific geographical regions which may eventually be vital for the most severely affected countries and regions for sharp development of custom-made vindication strategies.

Systematic characterization of mutations altering protein degradation in human cancers

The ubiquitin-proteasome system (UPS) is the primary route for selective protein degradation in human cells. The UPS is an attractive target for novel cancer therapies, but the precise UPS genes and substrates important for cancer growth are incompletely understood. Leveraging multi-omics data across more than 9,000 human tumors and 33 cancer types, we found that over 19% of all cancer driver genes affect UPS function. We implicate transcription factors as important substrates and show that c-Myc stability is modulated by CUL3. Moreover, we developed a deep learning model (deepDegron) to identify mutations that result in degron loss and experimentally validated the prediction that gain-of-function truncating mutations in GATA3 and PPM1D result in increased protein stability. Last, we identified UPS driver genes associated with prognosis and the tumor microenvironment. This study demonstrates the important role of UPS dysregulation in human cancer and underscores the potential therapeutic utility of targeting the UPS.

Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

AbstractThere is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α‐chymotrypsin (ChT), using a post‐fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i‐ChT) was distributed within the hydrogel. The enzyme‐immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i‐ChT activity, half‐life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i‐ChT loses activity compared to free ChT. Furthermore, enzyme half‐life increased up to seven‐fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i‐ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.

Protein Stability Perturbation Contributes to the Loss of Function in Haploinsufficient Genes.

Missense variants are among the most studied genome modifications as disease biomarkers. It has been shown that the “perturbation” of the protein stability upon a missense variant (in terms of absolute ΔΔG value, i.e., |ΔΔG|) has a significant, but not predictive, correlation with the pathogenicity of that variant. However, here we show that this correlation becomes significantly amplified in haploinsufficient genes. Moreover, the enrichment of pathogenic variants increases at the increasing protein stability perturbation value. These findings suggest that protein stability perturbation might be considered as a potential cofactor in diseases associated with haploinsufficient genes reporting missense variants.

Structure of the Complete Dimeric Human GDAP1 Core Domain Provides Insights into Ligand Binding and Clustering of Disease Mutations.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive toward classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point toward allosteric mechanisms in regulating GDAP1 oligomeric state and function.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction.

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Structural basis for the DNA-binding activity of human ARID4B Tudor domain

Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity. Structure determination and DNA titration analysis indicated that the ARID4B Tudor domain is also an interdigitated double Tudor domain with a DNA-binding surface similar to ARID4A. We identified a residue close to the DNA-binding site of the Tudor domain that differs between ARID4A and ARID4B. The Leu50 in ARID4A is Glu50 in ARID4B, and the latter forms salt bridges with two lysine residues at the DNA-binding surface. This causes a decrease in the strength of positive charge, thus reducing DNA-binding affinity while significantly increasing protein stability. We also found that a C-terminal extension region enhances the DNA-binding affinity of the ARID4B Tudor domain. This C-terminal extension is disordered and contains a positively charged RGR motif, providing an additional DNA-binding site. Finally, sequence and phylogenetic analyses indicated that the residue differences and the presence of the RGR extension region are conserved. These results provide new insight into the functional differences between ARID4A and ARID4B proteins, as well as elucidating the function of the disordered regions in these proteins.