Journal of biochemistry最新文献

Proteins are essential components in biotechnological and biopharmaceutical applications; however, their structural instability under alkaline conditions presents significant limitations. High-pH environments, such as chromatographic clean-in-place (CIP) protocols, frequently cause protein degradation and loss of biological activity. Current strategies for engineering alkali-stable proteins include rational design approaches targeting deamidation-susceptible residues, surface charge optimization, and enzyme extraction from alkaliphilic organisms. However, the fundamental principles governing alkaline stability remain poorly understood. In this study, we investigated alkaline stability mechanisms in Fc gamma receptor IIIa, a critical immune effector protein with applications in antibody purification and glycoform analysis. Systematic mutagenesis identified a tyrosine-to-phenylalanine substitution at position 59 that significantly enhanced protein stability during alkaline CIP exposure while retaining substantial IgG binding activity. Structural and biophysical characterizations revealed that this substitution prevents the deprotonation of tyrosine that occurs at alkaline pH, thereby mitigating destabilizing electrostatic repulsion within the protein structure. Our findings support a model in which targeted aromatic substitution enhances alkaline stability without severely compromising protein function and provide mechanistic insight into the contribution of buried tyrosine ionization to alkaline instability in FcγRIIIa.

Genetic and epigenetic alterations in colorectal cancer (CRC) have been extensively investigated. Small depressed-type colorectal tumors (DCs) constitute a highly invasive CRC subtype. We characterized the mutational profiles of 22 DC samples, seven of which were subjected to whole exome sequencing (WES). This revealed frequent mutations (7 of 22 cases, 31.8%) in PTPRK, with most of these mutations located in the D1 domain-encoding region. To further quantify the incidence of PTPRK mutations, we analyzed 567 CRCs cases from two public databases (The Cancer Genome Atlas and the Catalogue of Somatic Mutations in Cancer). Mutations were more frequently observed in proximal colon tumors and were highly associated with the CpG island methylator phenotype-positive (CIMP-P) subtype (76.2%) and microsatellite instability (MSI; 66.7%). PTPRK mutant proteins (particularly those with D1 domain) retained the ability to bind integrin beta-4 (ITGB4). In addition, CRC cells expressing PTPRK mutants exhibited increased ITGB4 phosphorylation, suggesting that the mutations impair the phosphatase activity of PTPRK. Consistent with this, tumors with PTPRK mutations proliferated significantly more rapidly than their wild type counterparts in vivo. These findings suggest that PTPRK mutations contribute to DC development by dysregulating phosphorylation-mediated signaling pathways.

Single-chain variable fragment (scFv) antibodies outperform conventional IgG antibodies due to their lower molecular weight, reduced immunogenicity, rapid clearance, and efficient bacterial expression. However, it remains difficult to achieve correct folding of scFv containing two intramolecular disulfide bonds in the reducing E. coli cytoplasm. We previously achieved correct folding of a trastuzumab-derived scFv by co-expressing the disulfide isomerase DsbC and the yeast sulfhydryl oxidase Erv1p in the cytoplasm of the E. coli SHuffle T7 strain. Here, we applied this system to produce the immune checkpoint inhibitor atezolizumab-derived scFv (denoted as atz-scFv). The atz-scFv was purified through a two-step chromatography process; however, insufficient disulfide bond formation led to contamination by proteins lacking a disulfide bonds. To address this, we developed and optimized a heat-treatment method in which atz-scFv was incubated at elevated temperatures to selectively precipitate protein species lacking a disulfide bond. The resulting atz-scFv exhibited increased apparent thermal stability and retained antigen-binding affinity compared to the untreated sample. This heat-treatment method offers a cost-effective alternative to traditional purification, eliminating the need for affinity chromatography while enhancing scFv stability and purity. The present results broaden scFv purification methods. (185 words).

Antibody engineering is often achieved through laborious mutagenesis and screening. However, the physicochemical basis of cross-reactivity-enhancing mutations remains unclear. We computationally redesigned the severe acute respiratory syndrome coronavirus (SARS-CoV)-1 neutralizing antibody m396 to recognize the SARS-CoV-2 receptor-binding domain (RBD) and characterized its biophysical properties. A first-generation variant carrying three light-chain substitutions (S30LW, S93LI, and S94LF) acquired detectable SARS-CoV-2 RBD binding, while strengthening its affinity for the SARS-CoV RBD. A second-generation variant carrying two substitutions (T52HL and L54HW) further improved SARS-CoV-2 binding with a low micromolar affinity, predominantly driven by an approximately 200-fold increase in the association rate. Circular dichroism spectra indicated preserved global folding across the variants, whereas differential scanning calorimetry revealed stepwise decreases in lower-temperature unfolding transitions. Hydrogen-deuterium exchange mass spectrometry showed increased dynamics of CDR-L1 and localized rigidification near CDR-H2 in the second variant. These results suggest a biophysical model in which a small number of mutations reprogram cross-recognition by redistributing the local conformational dynamics.

The expression of replication-dependent (RD) histones is tightly regulated by several pathways, including transcription and mRNA processing. However, the detailed mechanisms underlying the regulation of specific RD histone genes remain to be elucidated. Here, we demonstrated the differential responses of RD histone genes to severe hyperosmotic stress. Our data revealed an increased polyadenylation of the mRNA of some RD histones, although the RD histone mRNAs are known as to lack a poly(A) tail. Other RD histone genes showed elevated expression in total mRNA level. The nuclear bodies that mediate RD histone mRNA regulation, Cajal bodies, were disorganized after severe hyperosmotic stress. Depletion of the small nuclear RNA U7 (Rnu-7), which is involved in transcriptional suppression and processing of RD histone mRNAs, results in an increase in total mRNA levels with an identical specificity to hyperosmotic stress, but not polyadenylation. These findings demonstrated that RD histone genes were differentially regulated under normal conditions and in response to cellular stress.

L-Proline (L-Pro) and trans-4-hydroxy-L-proline (t-4-HPL) are major components of collagen and plant cell wall proteins. During their breakdown, free L-Pro and t-4-HPL are produced abundantly and play unique biological roles through complex metabolisms including isomerization. Therefore, it is essential to quantify all the 6 stereoisomers of proline and 4-hydroxyproline. Here, analytes were first labelled with 12C-dabsyl group, which has strong red color and stability under light. Second, each of the 13C6-dabsyl stereoisomers were added to 12C-dabsylated samples as internal standards before cleanup. Finally, the molar ratio of 12C- and 13C6-dabsyl analytes was determined as the intensity ratio of doublet molecular ions with m/z difference of 6 in mass spectra, which were obtained by subjecting the cleaned sample to capillary chromatography-mass spectrometry. The total amounts of proline, t-4-HP, and cis-4-hydroxyproline were determined using the standard addition method. Linear calibration curves (R2 > 0.99) were obtained in the range of 10-1000 pmol. To determine the molar ratio of D- and L-enantiomers, enantiopure dabsyl analyte fraction was obtained using reverse-phase and chiral chromatography. Mouse serum contained 0.20 μmol/L trans-4-hydroxy-D-proline. Achatina fulica hemolymph contained 0.070 μmol/L D-proline. The Santalum album wood powder contained all the 6 stereoisomers and unexpectedly the D-enantiomer was dominant for trans-4-hydroxyproline.

Aquatic invertebrates contain high levels of D-alanine in their tissues. D-Glutamate, a novel D-amino acid found in animal tissues, is exclusively found in the male reproductive tissues of the kuruma prawn Marsupenaeus japonicus. In this study, we investigated the effects of seasonal changes and high salinity seawater exposure on D- and L-glutamate and D- and L-alanine contents in male reproductive tissues of M. japonicus. We found that D-glutamate content was high during the breeding season specifically, D-glutamate content in the seminal receptacles was particularly high, suggesting that it is actively biosynthesized and supplied to the seminal receptacles for the regeneration of the spermatophores released during mating. The D- and L-alanine contents in the testes increased significantly after exposure to high-salinity seawater, whereas L-glutamate content did not change, and D-glutamate increased in some case, suggesting that the increase was due to factors related to reproductive function rather than their response to hyper-osmotic stimulation. This indicates that D-glutamate is crucial for the reproductive function of M. japonicus.

Microcompartments are miniaturized, uniform units designed to isolate and analyze individual cells, cell pairs, spheroids, or organoids, enabling massive parallel assays. This technique overcomes key limitations of traditional plate-based methods, including limited scale, high cost, and labor-intensive processing. This review highlights technologies that create these compartments, primarily leveraging droplet microfluidics and hydrogel techniques for high-throughput cellular analysis. These platforms are compatible with diverse measurement modalities, including imaging-based readouts for real-time monitoring, barcoded sequencing for multiplexed molecular profiling, and flow cytometry-based sorting for enriching functional populations. For example, water-in-oil droplets serve as picoliter-scale reaction vessels for rapid cell isolation based on antibody secretion profiles, while hydrogel platforms support single-cell clonal expansion for screening and the generation of uniform spheroids for drug testing. By enabling the massive parallelization of functional assays, microcompartment platforms are accelerating biomedical research and therapeutic development.

In mitochondria, the pyruvate dehydrogenase complex (PDHC) serves as a key metabolic regulator by converting glycolysis-derived pyruvate into acetyl-CoA, thereby controlling carbon flux into the tricarboxylic acid (TCA) cycle. PDHC activity is tightly regulated by two post-translational modifications: phosphorylation of the E1 subunit and lipoylation of the E2 subunit. While phosphorylation of E1 reversibly suppresses pyruvate dehydrogenase (PDH) activity, lipoylation of E2 is essential for intracomplex electron transfer reactions, and together these modifications define PDHC enzymatic activity. Mitochondrial respiratory supercomplexes (SCs) play a critical role in efficient electron transfer during mitochondrial respiration, and PDH has been reported to regulate SC organization. However, it remains unclear whether this regulatory mechanism, including subunit phosphorylation, is linked to protein lipoylation. In this study, we examined the impact of protein lipoylation on the phosphorylation status of the PDHC E1 subunit and on mitochondrial respiratory supercomplex formation during C2C12 differentiation. To this end, suppression of lipoic acid synthase (LIAS), a key enzyme responsible for mitochondrial protein lipoylation, in C2C12 cells resulted in dephosphorylation of the PDHC E1 subunit and formation of specific mitochondrial respiratory supercomplexes. These findings suggest that PDHC E1 dephosphorylation and specific mitochondrial respiratory supercomplex assembly can occur under conditions of impaired E2 lipoylation.

Innate immune receptors detect molecular features of pathogen presence and cellular damage, enabling cells to mount anti-microbial defenses including the secretion of proinflammatory cytokines. Though classically studied in immunocytes, a remarkably broad range of innate immune receptor activity is now recognized in adipocytes, including that of Toll-like receptors, NOD-like receptors, inflammasomes, and nucleic acid sensors such as cGAS-STING and RIG-I. These receptors influence adipocyte proinflammatory potential through control of secreted signaling factors that act in adipose tissue. Through less well understood mechanisms, they also influence adipocyte insulin sensitivity, lipolysis, fatty acid oxidation, and thermogenesis. Innate immune receptors are activated by a diverse array of stimuli including circulating signaling factors and intracellular metabolic stresses, especially those related to mitochondrial function. The receptors are thus a potential means by which obesity-associated signals act through adipocytes to drive inflammation, adipose dysfunction, and metabolic disease pathogenesis.