Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named \"Nigrospora oryzae mitovirus 3\" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.

Tardigrades are unique micro-organisms with a high tolerance to desiccation. The protection of their cells against desiccation involves tardigrade-specific proteins, which include the so-called cytoplasmic abundant heat soluble (CAHS) proteins. As a first step towards the design of peptides capable of mimicking the cytoprotective properties of CAHS proteins, we have synthesized several model peptides with sequences selected from conserved CAHS motifs and investigated to what extent they exhibit the desiccation-induced structural changes of the full-length proteins. Using circular dichroism spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations, we have found that the CAHS model peptides are mostly disordered, but adopt a more α $$ \\alpha $$ -helical structure upon addition of 2,2,2-trifluoroethanol, which mimics desiccation. This structural behavior is similar to that of full-length CAHS proteins, which also adopt more ordered conformations upon desiccation. We also have investigated the surface activity of the peptides at the air/water interface, which also mimics partial desiccation. Interestingly, sum-frequency generation spectroscopy shows that all model peptides are surface active and adopt a helical structure at the air/water interface. Our results suggest that amino acids with high helix-forming propensities might contribute to the propensity of these peptides to adopt a helical structure when fully or partially dehydrated. Thus, the selected sequences retain part of the CAHS structural behavior upon desiccation, and might be used as a basis for the design of new synthetic peptide-based cryoprotective materials.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.

Receptor-interacting protein kinase 1 (RIPK1) is a therapeutic target in treating neurodegenerative diseases and cancers. RIPK1 has three distinct functional domains, with the center domain containing a receptor-interacting protein homotypic interaction motif (RHIM), which mediates amyloid formation. The functional amyloid formed by RIPK1 and/or RIPK3 is a crucial intermediate in regulating cell necroptosis. In this study, the amyloid structure of mouse RIPK1, formed by an 82-residue sequence centered at RHIM, is presented. It reveals the \"N\"-shaped folding of the protein subunit in the fibril with four β-strands. The folding pattern is shared by several amyloid structures formed by proteins with RHIM, with the central β-strand formed by the most conserved tetrad sequence I/VQI/VG. However, the solid-state NMR results indicate a structural difference between mouse RIPK1 and mouse RIPK3. A change in the structural rigidity is also suggested by the observation of weakened signals for mouse RIPK3 upon mixing with RIPK1 to form the RIPK1/RIPK3 complex fibrils. Our results provide vital information to understand the interactions between different proteins with RHIM, which will help us further comprehend the regulation mechanism in cell necroptosis.

The identification of enzyme functions plays a crucial role in understanding the mechanisms of biological activities and advancing the development of life sciences. However, existing enzyme EC number prediction methods did not fully utilize protein sequence information and still had shortcomings in identification accuracy. To address this issue, we proposed an EC number prediction network using hierarchical features and global features (ECPN-HFGF). This method first utilized residual networks to extract generic features from protein sequences, and then employed hierarchical feature extraction modules and global feature extraction modules to further extract hierarchical and global features of protein sequences. Subsequently, the prediction results of both feature types were combined, and a multitask learning framework was utilized to achieve accurate prediction of enzyme EC numbers. Experimental results indicated that the ECPN-HFGF method performed best in the task of predicting EC numbers for protein sequences, achieving macro F1 and micro F1 scores of 95.5% and 99.0%, respectively. The ECPN-HFGF method effectively combined hierarchical and global features of protein sequences, allowing for rapid and accurate EC number prediction. Compared to current commonly used methods, this method offers significantly higher prediction accuracy, providing an efficient approach for the advancement of enzymology research and enzyme engineering applications.
酶功能的识别对理解生命活动的机制、推进生命科学的发展有重要作用。然而现有的酶EC编号预测方法，并未充分利用蛋白质序列信息，在识别精度上仍有所不足。针对上述问题，本研究提出一种基于层级特征和全局特征的EC编号预测网络(EC number prediction network using hierarchical features and global features, ECPN-HFGF)。该方法首先通过残差网络提取蛋白质序列通用特征，并通过层级特征提取模块和全局特征提取模块进一步提取蛋白质序列的层级特征和全局特征，之后结合两种特征信息的预测结果，采用多任务学习框架，实现酶EC编号的精确预测。计算实验结果表明，ECPN-HFGF方法在蛋白质序列EC编号预测任务上性能最佳，宏观F1值和微观F1值分别达到95.5%和99.0%。ECPN-HFGF方法能有效结合蛋白质序列的层级特征和全局特征，快速准确预测蛋白质序列EC编号，比当前常用方法预测精确度更高，能够为酶学研究和酶工程应用的发展提供一种高效的思路和方法。.

Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences. The model always ranks the binding of the cognate sequence in the top 100 sequences, and for 6 of the 8 mAbs, the cognate sequence ranks in the top ten. Practically, this approach has potential utility in selecting highly specific mAbs for therapeutics or diagnostics. More fundamentally, this demonstrates that very sparse random sampling of a large amino acid sequence spaces is sufficient to generate comprehensive models predictive of highly specific molecular recognition.

The process of protein phase separation into liquid condensates has been implicated in the formation of membraneless organelles (MLOs), which selectively concentrate biomolecules to perform essential cellular functions. Although the importance of this process in health and disease is increasingly recognized, the experimental identification of proteins forming MLOs remains a complex challenge. In this study, we addressed this problem by harnessing the power of AlphaFold2 to perform computational predictions of the conformational properties of proteins from their amino acid sequences. We thus developed the CoDropleT (co-condensation into droplet transformer) method of predicting the propensity of co-condensation of protein pairs. The method was trained by combining experimental datasets of co-condensing proteins from the CD-CODE database with curated negative datasets of non-co-condensing proteins. To illustrate the performance of the method, we applied it to estimate the propensity of proteins to co-condense into MLOs. Our results suggest that CoDropleT could facilitate functional and therapeutic studies on protein condensation by predicting the composition of protein condensates.

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

In this work, we explored the intrinsic disorder status of the three members of the synuclein family of proteins-α-, β-, and γ-synucleins-and showed that although all three human synucleins are highly disordered, the highest levels of disorder are observed in γ-synuclein. Our analysis of the peculiarities of the amino acid sequences and modeled 3D structures of the human synuclein family members revealed that the pathological mutations A30P, E46K, H50Q, A53T, and A53E associated with the early onset of Parkinson\'s disease caused some increase in the local disorder propensity of human α-synuclein. A comparative sequence-based analysis of the synuclein proteins from various evolutionary distant species and evaluation of their levels of intrinsic disorder using a set of commonly used bioinformatics tools revealed that, irrespective of their origin, all members of the synuclein family analyzed in this study were predicted to be highly disordered proteins, indicating that their intrinsically disordered nature represents an evolutionary conserved and therefore functionally important feature. A detailed functional disorder analysis of the proteins in the interactomes of the human synuclein family members utilizing a set of commonly used disorder analysis tools showed that the human α-synuclein interactome has relatively higher levels of intrinsic disorder as compared with the interactomes of human β- and γ- synucleins and revealed that, relative to the β- and γ-synuclein interactomes, α-synuclein interactors are involved in a much broader spectrum of highly diversified functional pathways. Although proteins interacting with three human synucleins were characterized by highly diversified functionalities, this analysis also revealed that the interactors of three human synucleins were involved in three common functional pathways, such as the synaptic vesicle cycle, serotonergic synapse, and retrograde endocannabinoid signaling. Taken together, these observations highlight the critical importance of the intrinsic disorder of human synucleins and their interactors in various neuronal processes.