Biochimica et biophysica acta. Proteins and proteomics最新文献_第2页

Enhancement of the nucleotide incorporation activity of the terminal deoxynucleotidyl transferase by N-terminal truncation and DNA-binding protein modulation 通过n端截断和dna结合蛋白调节增强末端脱氧核苷酸转移酶的核苷酸结合活性。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-12 DOI: 10.1016/j.bbapap.2025.141114

Antos B. Sachanka, Stafaniya S. Douhaya, Veronika V. Shchur, Aliaksei V. Yantsevich

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that catalyzes the template-independent addition of nucleotides to the 3′ terminus of single-stranded DNA. Its distinctive catalytic properties have been exploited in aptasensor design, nanomaterial synthesis, DNA mutagenesis, innovative data storage based on single-nucleotide DNA sequences, and enzymatic de novo DNA synthesis. However, the application of the enzyme is limited by its low expression yield, poor thermostability, and reduced nucleotide incorporation efficiency with substrates prone to forming secondary structures. To overcome these limitations, we engineered a bovine TdT variant truncated at the N-terminus by 148 residues (148_TrTdT), which demonstrates a twofold increase in expression yield, a 5 °C improvement in thermostability, and over 30 % enhancement in nucleotide incorporation efficiency and processivity compared to the wild-type enzyme. Moreover, we demonstrated that DNA-binding proteins enhance the nucleotide incorporation activity of native TdT (10-fold) and 148_TrTdT (2-fold) when substrates are prone to forming secondary structures. Collectively, the methodologies implemented in this study exhibit significant potential for enhancing the efficiency of enzymatic de novo DNA synthesis, thereby enabling the development of new bioengineering and biomedical applications.

末端脱氧核苷酸转移酶（TdT）是一种独特的DNA聚合酶，它催化核苷酸在单链DNA 3'端独立于模板的添加。其独特的催化特性已被用于配体传感器设计、纳米材料合成、DNA诱变、基于单核苷酸DNA序列的创新数据存储和酶促从头合成DNA。然而，该酶的应用受到其表达量低、热稳定性差以及与底物形成二级结构的核苷酸结合效率降低的限制。为了克服这些限制，我们设计了一个在n端被148个残基截断的牛TdT变体（148_TrTdT），与野生型酶相比，其表达量增加了两倍，热稳定性提高了5 °C，核苷酸结合效率和加工效率提高了30 %以上。此外，我们还证明，当底物易于形成二级结构时，dna结合蛋白可增强天然TdT（10倍）和148_TrTdT（2倍）的核苷酸结合活性。总的来说，本研究中实施的方法在提高酶促从头DNA合成的效率方面表现出巨大的潜力，从而使新的生物工程和生物医学应用得以发展。

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引用次数: 0

Integrated computational and biological assays to identify and validate L. donovani homoserine dehydrogenase inhibitors 综合计算和生物测定鉴定和验证L. donovani高丝氨酸脱氢酶抑制剂。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-11 DOI: 10.1016/j.bbapap.2025.141112

Shalu Verma , Shaswat Sengar , Abdul Basit Khan , Anil Kumar Shakya , J. Venkatesh Pratap

The Neglected Tropical Disease (NTD) Leishmaniasis continues to pose a serious global health burden affecting millions globally. Visceral leishmaniasis, one of three clinical forms, can be fatal, if left untreated. Current treatment regimen relies mostly on re-purposed drugs and is limited by toxicity, high cost and the emergence of drug resistance, highlighting the need for safer, targeted therapies. In this study, we characterized Leishmania donovani homoserine dehydrogenase (LdHSD), a crucial enzyme in the aspartate pathway. As no human homolog exists, it represents a promising target for selective therapeutic intervention. LdHSD was purified to homogeneity, characterized biophysically and its activity biochemically assayed. Docking of the substrate (L-homoserine) and the cofactor (NADP⁺) to the LdHSD homology model identified adjacent but discrete binding pockets. Structure-based virtual screening of the Maybridge Compound Library was subsequently performed and the top 10 substrate-site bound compounds were taken for experimental validation. Two of the three piperidine/piperazine scaffold compounds inhibited LdHSD activity by ∼90 %. These findings highlight the therapeutic potential of targeting LdHSD for the development of novel, selective anti-leishmanial agents and establish a strong foundation for future optimization of these identified inhibitors.

被忽视的热带病利什曼病继续构成严重的全球健康负担，影响全球数百万人。内脏利什曼病是三种临床形式之一，如果不及时治疗，可能会致命。目前的治疗方案主要依赖于重新利用的药物，受到毒性、高成本和耐药性出现的限制，突出表明需要更安全的靶向治疗。在这项研究中，我们鉴定了利什曼原虫同型丝氨酸脱氢酶（LdHSD），这是天冬氨酸途径中的一个关键酶。由于没有人类同源物存在，它代表了选择性治疗干预的一个有希望的目标。对LdHSD进行纯化至均匀性，进行生物物理表征，并进行生物化学活性测定。底物（l -高丝氨酸）和辅因子（NADP）对接到LdHSD同源模型，确定了相邻但离散的结合口袋。随后对Maybridge化合物库进行基于结构的虚拟筛选，并选取前10个底物-位点结合化合物进行实验验证。三种哌啶/哌嗪支架化合物中的两种抑制LdHSD活性~90 %。这些发现突出了以LdHSD为靶点开发新型、选择性抗利什曼药物的治疗潜力，并为这些已确定的抑制剂的未来优化奠定了坚实的基础。

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引用次数: 0

Integrative multi-omics machine learning reveals novel driver genes associations in lung adenocarcinoma 综合多组学机器学习揭示了肺腺癌中新的驱动基因关联。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-08 DOI: 10.1016/j.bbapap.2025.141113

Fei Yuan , FeiMing Huang , Xiaoyu Cao , Yu-Hang Zhang , KaiYan Feng , YuSheng Bao , Tao Huang , Yu-Dong Cai

Lung adenocarcinoma remains a major challenge in cancer research due to its complex molecular underpinnings. In this study, we developed an integrated machine learning framework to identify novel driver genes associated with lung adenocarcinoma by leveraging multi-omics data. We curated gene candidates from methylation, RNAseq, mutation, and miRNA levels, and mapped them onto a protein–protein interaction network from STRING to generate informative feature vectors using a node2vec method. Furthermore, they were also represented by GO and KEGG enrichment features. All features were then refined through a multi-step process, beginning with the Boruta algorithm for filtering and followed by the minimum redundancy maximum relevance method for ranking. An incremental feature selection strategy was employed to determine the optimal feature subsets, which were used to build predictive models with random forest and support vector machine classifiers. To address class imbalance, synthetic sampling was applied, and ten-fold cross-validation ensured model robustness. Consequently, we predicted 428, 105, 1039, and 1748 potential lung adenocarcinoma driver genes for RNAseq, methylation, mutation, and miRNA levels, respectively. Integrated analysis of overlapping gene sets further highlighted key candidates, including PQLC3, FAM192A, FAM83D, SPRED1, SFTPB, and TM4SF5, with high composite probability scores. Some identified genes may be the driver genes of lung adenocarcinoma and have some druggable potential. These findings provide new insights into the molecular mechanisms of lung adenocarcinoma and suggest promising targets for future diagnostic and therapeutic strategies.

由于其复杂的分子基础，肺腺癌仍然是癌症研究的主要挑战。在这项研究中，我们开发了一个集成的机器学习框架，通过利用多组学数据来识别与肺腺癌相关的新驱动基因。我们筛选了来自甲基化、RNAseq、突变和miRNA水平的候选基因，并将它们映射到来自STRING的蛋白质-蛋白质相互作用网络上，使用node2vec方法生成信息丰富的特征向量。此外，它们还具有GO和KEGG富集特征。然后通过多步骤过程对所有特征进行细化，首先使用Boruta算法进行过滤，然后使用最小冗余最大相关性方法进行排序。采用增量特征选择策略确定最优特征子集，并结合随机森林和支持向量机分类器构建预测模型。为了解决类别不平衡问题，采用了合成抽样，并进行了十倍交叉验证，确保了模型的稳健性。因此，我们分别预测了428、105、1039和1748个潜在的肺腺癌驱动基因的RNAseq、甲基化、突变和miRNA水平。对重叠基因集的综合分析进一步突出了关键候选基因，包括PQLC3、FAM192A、FAM83D、SPRED1、SFTPB和TM4SF5，它们的复合概率得分很高。一些已鉴定的基因可能是肺腺癌的驱动基因，具有一定的药物潜力。这些发现为肺腺癌的分子机制提供了新的见解，并为未来的诊断和治疗策略提供了有希望的靶点。

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引用次数: 0

Novel interactions between FOXP2 and PAX6: Implications for neural development and autism spectrum disorders FOXP2和PAX6之间新的相互作用：对神经发育和自闭症谱系障碍的影响。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-08 DOI: 10.1016/j.bbapap.2025.141110

Jessica Brothwell , Olamide Jeje , Dineo Mashamaite , Sylvia Fanucchi

FOXP2 and PAX6 are transcription factors essential for neural development, with mutations in both linked to autism spectrum disorders (ASDs). Their DNA-binding domains include a forkhead domain (FHD) for FOXP2 and a paired domain (PD) plus homeodomain (HD) for PAX6. We investigated whether the FOXP2 FHD interacts directly with PAX6 PD or HD, and how such interactions influence DNA binding. Fluorescence anisotropy showed that all three domains bind specifically to their respective DNA targets with similar affinities. The FOXP2 FHD also interacts directly with both PAX6 PD and HD, with low micromolar binding affinities. Despite its stronger intrinsic DNA affinity, the FHD was displaced from its target DNA by both PAX6 domains, suggesting that protein–protein interactions can override DNA affinity under competitive conditions. In contrast, FOXP2 could not displace PD or HD from their DNA targets. Molecular docking supported these findings: DNA–protein interfaces were largely unchanged by the second protein, but protein–protein interfaces were strongly influenced by DNA occupancy. The H3 helix of FHD was identified as a central point for assembly, contributing to both DNA and protein interfaces. When FHD was bound to DNA, H3 was occupied, forcing PD or HD to dock at alternative, less optimal sites. HD maintained stronger contacts in these rearranged states, consistent with its greater competitive strength. This asymmetric interplay indicates competitive dominance by PAX6 and suggests mechanisms that could underlie transcriptional regulation in neurodevelopment.

FOXP2和PAX6是神经发育所必需的转录因子，两者的突变都与自闭症谱系障碍（asd）有关。它们的dna结合域包括FOXP2的叉头结构域（FHD）和PAX6的成对结构域（PD）加同源结构域（HD）。我们研究了FOXP2 FHD是否直接与PAX6 PD或HD相互作用，以及这种相互作用如何影响DNA结合。荧光各向异性表明，这三个结构域特异性结合各自的DNA目标具有相似的亲和力。FOXP2 FHD也直接与PAX6 PD和HD相互作用，具有低微摩尔结合亲和力。尽管FHD具有更强的内在DNA亲和力，但它被两个PAX6结构域从目标DNA中取代，这表明在竞争条件下，蛋白质-蛋白质相互作用可以覆盖DNA亲和力。相比之下，FOXP2不能将PD或HD从它们的DNA靶标上取代。分子对接支持这些发现：DNA-蛋白质界面在很大程度上不受第二个蛋白质的影响，但蛋白质-蛋白质界面受到DNA占用的强烈影响。FHD的H3螺旋被确定为组装的中心点，有助于DNA和蛋白质的界面。当FHD与DNA结合时，H3被占用，迫使PD或HD停靠在替代的、不太理想的位置。HD在这些重组后的州保持了更强的联系，这与它更强的竞争实力相一致。这种不对称的相互作用表明PAX6具有竞争性优势，并提示了神经发育中转录调节的机制。

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引用次数: 0

Biophysical characterization of Hpa1 protein from Xanthomonas orzyae 黄单胞菌Hpa1蛋白的生物物理特性研究。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-07 DOI: 10.1016/j.bbapap.2025.141111

Jaimini Patoliya , Khushali Thaker , Jahanvi Bajpai , Dhaval Patel , Nayan Jain , Prasant Kumar , Rushikesh Joshi

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight in rice, secretes a suite of effectors via the Type III Secretion System (T3SS), among which harpin proteins like Hpa1 are key modulators of plant immunity. Unlike other T3SS effectors, Hpa1 lacks enzymatic activity and instead acts as a biostimulant, promoting growth and defense responses. In this study, the hpa1 gene was cloned from Xoo BXO1 strain and successfully expressed in E. coli Rosetta cells. The recombinant protein was purified using Ni-NTA chromatography, and SDS-PAGE, Western blotting, and MALDI-TOF analysis confirmed its molecular weight (∼16.5 kDa). Biophysical characterization revealed a predominance of α-helical content, with CD spectroscopy. Thermal and chemical unfolding of Hpa1 was evaluated using CD spectroscopy (with GuHCl) and ANS fluorescence (with GuHCl and urea), revealing moderate stability. pH-dependent stability assessed by fluorescence showed optimal structural retention at pH 5. TFE-induced structural modulation, studied by CD, demonstrated concentration-dependent α-helix enhancement. Thermal stability was assessed through four approaches: SDS-PAGE, CD at 222 nm, SYPRO Orange-based thermal shift assay, and HR-inducing activity in Nicotiana benthamiana. These results collectively confirmed that native Hpa1 conformation is not necessary for its function. These findings highlight its potential as a protein-based biostimulant for agricultural applications and its utility as a model for studying intrinsically disordered yet functionally stable proteins.

米黄单胞菌。水稻白叶枯病病原菌oryzae （Xoo）通过III型分泌系统（Type III分泌系统，T3SS）分泌一系列效应物，其中Hpa1等harpin蛋白是植物免疫的关键调节剂。与其他T3SS效应物不同，Hpa1缺乏酶活性，而是作为生物刺激物，促进生长和防御反应。本研究从Xoo BXO1菌株中克隆了hpa1基因，并在大肠杆菌Rosetta细胞中成功表达。重组蛋白经Ni-NTA层析纯化，SDS-PAGE、Western blotting和MALDI-TOF分析证实其分子量为~16.5 kDa。生物物理表征表明α-螺旋含量占主导地位。利用CD光谱（含GuHCl）和ANS荧光（含GuHCl和尿素）对Hpa1的热展开和化学展开进行了评估，结果显示Hpa1的稳定性中等。荧光测定的pH依赖性稳定性表明，pH为 5时结构保持最佳。CD研究了tfe诱导的结构调制，显示出浓度依赖性α-螺旋增强。通过四种方法评估热稳定性：SDS-PAGE、222 nm CD、SYPRO橙基热移法和benthamiana诱导hr活性。这些结果共同证实了天然Hpa1构象对其功能不是必需的。这些发现突出了它作为一种基于蛋白质的生物刺激素在农业应用中的潜力，以及它作为研究内在无序但功能稳定的蛋白质的模型的效用。

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引用次数: 0

Self-association of Apolipoprotein A-I studied with multiple-association models: A pyrene multiparametric analysis. 多关联模型研究载脂蛋白A- i的自关联：芘多参数分析。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-11-01 Epub Date: 2025-08-06 DOI: 10.1016/j.bbapap.2025.141093

Wilson A Tárraga, Lisandro J Falomir-Lockhart, Horacio A Garda, Marina C Gonzalez

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein particles, conferring anti-atherogenic properties through reverse cholesterol transport. In its lipid-free state, apoA-I self-associates, a process implicated in normal physiology as well as in pathological conditions, such as amyloidosis. Although various mechanisms have been proposed to explain its self-association, there is still no consensus, and its functional implications remain unclear. Here, we employed a multi-parametric fluorescent probe to investigate apoA-I self-association. We used three single cysteine mutants located in different helixes: K107C (H4), K133C (H5), and F225C (H10); and labelled them with pyrene as detailed previously (Tárraga et al. Arch Biochem Biophys 699 (2021) 108748). Our original experiments revealed excimer emission between helixes H5 and H10 and polarity changes also in H4. By exploiting specific pyrene band emissions to monitor dimer association (via excimer formation) and microenvironment polarity (P-value), we tracked apoA-I oligomerization as a function of protein concentration. Several mathematical models of self-association were developed and compared using selection criteria to identify the simplest model reproducing apoA-I's complex behaviour in aqueous media: a Sequential Association submodel limited to a tetramer as the highest order oligomeric species and considering both dimers and tetramers as responsible for the excimer's emission. Multi-equilibria models enhanced the titration analysis, allowing estimation of association constants (Ka) and oligomeric species distribution. Our results support previous evidence that contacts among helixes, which stabilize discoidal HDL particles, are already present in lipid-free apoA-I, with dimeric species predominating, and possible tetrameric too. Further investigation of these species is essential to elucidate their physiological and pathological roles, such as in atherosclerosis.

载脂蛋白A-I （apoA-I）是高密度脂蛋白颗粒的主要蛋白，通过逆向胆固醇转运具有抗动脉粥样硬化特性。在无脂状态下，apoa - 1自我结合，这一过程涉及正常生理和病理条件，如淀粉样变性。尽管人们提出了各种机制来解释其自我关联，但仍未达成共识，其功能含义仍不清楚。在这里，我们采用多参数荧光探针来研究apoA-I的自关联。我们使用了三个位于不同螺旋上的单半胱氨酸突变体：K107C （H4）、K133C （H5）和F225C (H10)；如前所述，用芘标记它们（Tárraga等）。生物化学学报，699(2021)108748。我们最初的实验发现H5和H10螺旋之间的准分子发射和H4的极性也发生了变化。通过利用特定的芘带发射来监测二聚体结合（通过准分子形成）和微环境极性（p值），我们跟踪了apoA-I寡聚化作为蛋白质浓度的函数。研究人员开发了几种自结合的数学模型，并使用选择标准进行了比较，以确定再现apoA-I在水介质中复杂行为的最简单模型：一个顺序结合子模型，该模型将四聚体作为最高阶寡聚物，并考虑到二聚体和四聚体都负责准分子的发射。多平衡模型增强了滴定分析，允许估计缔合常数（Ka）和低聚物种分布。我们的研究结果支持了先前的证据，即在无脂apoa - 1中已经存在稳定盘状HDL颗粒的螺旋之间的接触，二聚体占主导地位，也可能是四聚体。进一步研究这些物种对于阐明其生理和病理作用至关重要，例如动脉粥样硬化。

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引用次数: 0

Drug–biomolecular interaction: Spectroscopic and computational insights into esomeprazole binding with human serum albumin 药物-生物分子相互作用：埃索美拉唑与人血清白蛋白结合的光谱和计算见解。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-10-31 DOI: 10.1016/j.bbapap.2025.141109

Anna Tanuja Safala Bodapati , Ragaiahgari Srinivas Reddy , Kandikonda Lavanya , Shravya Rao Madku , Rajdeep Chowdhury , Bijaya Ketan Sahoo

Cellular process relies on specific binding of drug molecules with desired biological receptors. Biomolecular receptors and drugs exhibit their biological functions upon their interactions. Understanding the binding parameters and energetics of the binding provides a plethora of information that may be helpful in drug design and discovery. Further, drug interaction with carrier proteins affects the pharmacokinetics and dynamics of other exogenous and endogenous drugs. In this work, we report the binding behavior of esomeprazole with human serum albumin using several biophysical techniques. Qualitative and quantitative aspects of the binding along with the binding energetics has been focused in extracting the thermodynamics parameters and key interaction force. The binding constants (K_b)obtained from Scatchard analysis are-1.17 × 10⁵, 7.49 × 10⁴, and 3.23 × 10⁴ M⁻¹ at 298, 303, 308 K respectively. Esomeprazole binds preferentially at site 1 in subdomain IIA of human serum albumin and was confirmed, supported by site-specific marker displacement studies and docking simulations. Negative value of change in free energy (∆G⁰) -32 kJ/mol at 298 K showed thermodynamically favourable interaction and outweigh of entropic factor (T∆S⁰ = 230.76 ± 3 kJ for T = 298 K) over the enthalpic contribution (∆H⁰ = −261.99 kJ/mol) revealed an entropy-driven process. Binding of esomeprazole affected the helical structure of human serum albumin. Molecular docking studies (theoretical) shows the binding pocket of ESM at site1(IIA), which is in accordance with the experimental result. Further, the interface residues involved in the binding were analysed from the 2D diagram and ligplot of the docked complex.

细胞过程依赖于药物分子与所需生物受体的特异性结合。生物分子受体与药物通过相互作用发挥其生物学功能。了解结合参数和结合的能量学提供了大量的信息，可能有助于药物设计和发现。此外，药物与载体蛋白的相互作用会影响其他外源性和内源性药物的药代动力学和动力学。在这项工作中，我们使用几种生物物理技术报道了埃索美拉唑与人血清白蛋白的结合行为。结合热力学参数和关键作用力的提取，从定性和定量两个方面进行了研究。Scatchard分析得到的结合常数（Kb）在298、303、308 K处分别为1.17 × 105、7.49 × 104和3.23 × 104 M-1。埃索美拉唑优先结合人血清白蛋白IIA亚结构域的1号位点，这一发现得到了位点特异性标记位移研究和对接模拟的证实。负值自由能的变化(∆G0) -32 焦每摩尔298 K显示热力学有利的互动和大于熵的因子(T∆S0 = 230.76 ± 3 kJ 298 T =  K)在向左反应贡献(∆H0 = -261.99 焦每摩尔)显示一个熵驱动过程。埃索美拉唑的结合影响了人血清白蛋白的螺旋结构。分子对接研究（理论）显示ESM的结合袋位于site1(IIA)，与实验结果一致。此外，通过对接配合物的二维图和光图分析了参与结合的界面残基。

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引用次数: 0

Multifunctionality analysis of serine hydroxymethyltransferases from human and Escherichia coli 人和大肠杆菌丝氨酸羟甲基转移酶的多功能分析

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-10-25 DOI: 10.1016/j.bbapap.2025.141107

Mahiro Hayashi , Kumiko Sakai-Kato , Tetsuya Miyamoto

Serine hydroxymethyltransferase (SHMT) catalyzes the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In a previous study, we found that human and Escherichia coli SHMTs possess THF-dependent d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. Some activities including aldolase and racemase activities have also been reported for SHMTs. In the present study, we investigated the multifunctionality of E. coli SHMT and two human SHMTs. All three SHMTs displayed high aldolase activity toward l-allo-threonine and l-threo-phenylserine, and measurable activity toward l-threonine, but they did not act on d-allo-threonine and d-threonine. The catalytic efficiency (k_cat/K_m) for l-allo-threonine was higher than for l-threo-phenylserine. None of the SHMTs displayed racemase activity toward various amino acids, although slight alanine racemase activity was detected for E. coli SHMT. Likewise, none of the SHMTs showed lyase, aminotransferase, or aspartate decarboxylase activities, and none exhibited dehydratase activity toward other hydroxy amino acids except d-serine. SHMTs are multifunctional enzymes possessing canonical hydroxymethyltransferase, d-serine dehydratase, and low-specificity l-threonine aldolase activities.

丝氨酸羟甲基转移酶（SHMT）催化l-丝氨酸和四氢叶酸（THF）分别转化为甘氨酸和5,10-亚甲基四氢叶酸。在之前的研究中，我们发现人类和大肠杆菌的SHMTs具有thf依赖性d-丝氨酸脱水酶活性，可将d-丝氨酸降解为丙酮酸和氨。一些活性，包括醛缩酶和消旋酶活性也被报道为SHMTs。在本研究中，我们研究了大肠杆菌SHMT和两种人类SHMT的多功能性。所有三种shmt对l-异体苏氨酸和l-三苯基丝氨酸均表现出较高的醛缩酶活性，对l-苏氨酸具有可测量的活性，但对d-异体苏氨酸和d-苏氨酸没有作用。l-异丙苏氨酸的催化效率（kcat/Km）高于l-三苯基丝氨酸。虽然在大肠杆菌SHMT中检测到轻微的丙氨酸外消旋酶活性，但没有一种SHMT显示出对各种氨基酸的外消旋酶活性。同样，没有一个SHMTs显示裂解酶、转氨酶或天冬氨酸脱羧酶活性，也没有显示除d-丝氨酸外的其他羟基氨基酸的脱水酶活性。SHMTs是多功能酶，具有典型的羟甲基转移酶、d-丝氨酸脱水酶和低特异性的l-苏氨酸醛缩酶活性。

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引用次数: 0

Identification of new protein-coding potential in Leishmania braziliensis using a proteogenomics approach 利用蛋白质基因组学方法鉴定巴西利什曼原虫新的蛋白质编码潜力

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-10-25 DOI: 10.1016/j.bbapap.2025.141108

Aditi Shenoy , Soumi Chowdhury , Shubhankar Pawar , Nitin Tupperwar , Harsh Pawar

American tegumentary leishmaniasis (ATL) is primarily caused by Leishmania (Viannia) species such as Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, which show complex genomic organisation and stage-specific adaptations underlying their pathogenicity. Despite the availability of its reference genome, limitations in gene annotation persist due to the presence of hypothetical proteins, pseudogenes, and unrecognised coding regions. In this study, we used a proteogenomic approach integrating publicly available high-resolution mass spectrometry data with a custom six-frame translated genome database to refine the genome annotation of L. braziliensis strain MHOM/BR/75/M2904. Utilising stringent database-dependent searches with a 1 % false discovery rate, we identified many unique peptides, of which 1034 were genome search-specific peptides (GSSPs) mapping exclusively to unannotated genomic regions. These GSSPs facilitated the discovery of 56 novel protein-coding genes and the correction of 228 existing gene models, including N- and C-terminal extensions. Notably, several novel genes encode proteins with conserved domains such as membrane attack complex/perforin (MACPF), kinesin K39, and peptidase S9/S15, suggesting functional relevance in parasite biology. Our findings demonstrate the power of proteogenomics to uncover cryptic protein-coding regions and improve genome annotations beyond conventional predictions. This refined annotation enhances our understanding of L. braziliensis biology, providing a more accurate proteomic landscape that can inform studies on parasite virulence, host interaction, and potential therapeutic targets. The study underscores the importance of integrating proteomic evidence with genomic data to capture the full coding potential of kinetoplastid parasites, paving the way for improved diagnostics and interventions against leishmaniasis.

美洲土著利什曼病（ATL）主要由巴西利什曼原虫、巴拿马利什曼原虫和圭亚那利什曼原虫等利什曼原虫引起，它们表现出复杂的基因组组织和阶段特异性适应，是其致病性的基础。尽管有参考基因组的可用性，但由于存在假设的蛋白质、假基因和未识别的编码区，基因注释的局限性仍然存在。在这项研究中，我们使用蛋白质基因组学方法整合公开的高分辨率质谱数据和定制的六帧翻译基因组数据库来完善巴西乳杆菌菌株MHOM/BR/75/M2904的基因组注释。利用严格的数据库依赖搜索（错误发现率为1%），我们鉴定出许多独特的肽，其中1034个是基因组搜索特异性肽（gssp），仅映射到未注释的基因组区域。这些gssp促进了56个新的蛋白质编码基因的发现，并纠正了228个现有的基因模型，包括N端和c端扩展。值得注意的是，一些新基因编码具有保守结构域的蛋白质，如膜攻击复合物/穿孔素（MACPF）、激酶蛋白K39和肽酶S9/S15，这表明它们在寄生虫生物学中具有功能相关性。我们的研究结果证明了蛋白质基因组学在揭示隐蛋白编码区和改进基因组注释方面的能力，超出了传统的预测。这一改进的注释增强了我们对巴西乳杆菌生物学的理解，提供了一个更准确的蛋白质组学景观，可以为寄生虫毒力、宿主相互作用和潜在治疗靶点的研究提供信息。这项研究强调了将蛋白质组学证据与基因组数据结合起来的重要性，以捕获着丝质体寄生虫的全部编码潜力，为改进利什曼病的诊断和干预措施铺平道路。

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引用次数: 0

AttnSeq-PPI: Enhancing protein-protein interaction network prediction using transfer learning-driven hybrid attention AttnSeq-PPI：利用迁移学习驱动的混合注意增强蛋白质相互作用网络预测。

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Proteins and proteomics

Pub Date : 2025-10-24 DOI: 10.1016/j.bbapap.2025.141102

Dipayan Sarkar, Chiranjib Sarkar

Study of protein-protein interaction (PPI) network is fundamental to all cellular processes in living organisms. PPI study based on experimental techniques such as high-throughput assays, mass spectrometry is time-consuming and expensive. Computational techniques like molecular docking are restricted to availability of 3D protein structures and not suitable for large numbers of proteins. On the contrary Sequence-based PPI study is advantageous over the limitations of above techniques. Sequence-based PPI study has gained a broader scope with the help of deep learning techniques and large language model (LLM). In this study, we proposed AttnSeq-PPI, a deep learning framework based on two channels of hybrid attention mechanism. The protein sequences are embedded in high dimensional space using ProtT5 language model. In our model hybrid attention mechanism is designed by combining self-attention and cross-attention which enable the model to extract features from each protein with respect to the contextual features of both the proteins. The hybrid attention mechanism effectively captures long-range dependencies within protein sequences as well as interacting features of both proteins. The model was trained and evaluated based on 5-fold cross validation on intra-species and multi-species datasets. Additionally, four datasets of independent species and true PPI network datasets were used for validation. AttnSeq-PPI demonstrates superior generalization and outperforms existing models, achieving 99 % accuracy for both human and multi-species datasets. It can provide prediction of novel PPIs with fewer false negatives and higher precision. Additionally, we developed a web-based tool on AttnSeq-PPI accessible at https://compbiosysnbu.in/attnseqppi/ to provide PPI prediction based on protein sequences.

蛋白质-蛋白质相互作用（PPI）网络的研究是生物体中所有细胞过程的基础。基于高通量分析、质谱等实验技术的PPI研究耗时且昂贵。像分子对接这样的计算技术仅限于三维蛋白质结构的可用性，不适合大量蛋白质。相反，基于序列的PPI研究优于上述技术的局限性。在深度学习技术和大型语言模型（LLM）的帮助下，基于序列的PPI研究获得了更广泛的范围。在本研究中，我们提出了一种基于双通道混合注意机制的深度学习框架AttnSeq-PPI。利用ProtT5语言模型将蛋白质序列嵌入到高维空间中。在我们的模型中，混合注意机制通过结合自注意和交叉注意来设计，使模型能够根据两种蛋白质的上下文特征从每种蛋白质中提取特征。混合注意机制有效地捕获了蛋白质序列内的长期依赖关系以及两种蛋白质的相互作用特征。基于种内和多物种数据集的5倍交叉验证对模型进行了训练和评估。此外，使用4个独立物种数据集和真实PPI网络数据集进行验证。AttnSeq-PPI表现出卓越的泛化能力，优于现有模型，在人类和多物种数据集上都达到了99% %的准确率。该方法对新型ppi的预测具有较低的假阴性和较高的预测精度。此外，我们开发了一个基于web的AttnSeq-PPI工具，可访问https://compbiosysnbu.in/attnseqppi/，以提供基于蛋白质序列的PPI预测。

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引用次数: 0