Evolutionary Analysis and Comparative Genomics of Protein Superfamilies

真核生物的起源以几种新的亚细胞系统的出现为标志。其中之一是内质网的钙（Ca 2+）储存系统，它深刻地影响细胞功能的各个方面，包括信号转导，运动，分裂和生物矿化。我们使用比较基因组学以及敏感的序列和结构分析来研究该系统的进化。我们的研究结果重建了最后一个真核生物共同祖先中Ca 2+储存系统的核心形式，因为至少有15种蛋白质构成了促进Ca 2+跨内膜流动和Ca 2+依赖性信号传导的基本系统。我们发现，EF-手钙离子结合的关键组件有其起源于一个可能的细菌共生体以外的线粒体祖细胞，而蛋白磷酸酶亚基的祖先钙调神经磷酸酶复合物很可能是从asgardarchaeal祖先的干真核生物继承。这进一步指出了真核生物在富含Ca 2+的生物矿化环境中的潜在起源，如基质层。我们进一步表明，在整个真核生物的进化有几个收购从细菌的关键组成部分的Ca 2 +-商店系统，即使没有原核谱系拥有一个可比的系统。此外，使用来自比较基因组学的定量测量，我们发现，有几轮的谱系特异性基因扩增，创新的新基因家族，和基因丢失与生物创新，如生物矿化的软体动物壳，颗石藻，和动物运动。动物新基因创新的爆发包括与人类Wolfram综合征相关的Wolfram蛋白。我们首次发现它包含以前未鉴定的Sel 1，EF-手和OB-折叠结构域，这可能在其生物化学中起关键作用。 HMCES（5 hmC结合，胚胎干细胞特异性蛋白）最初被鉴定为能够结合5-羟甲基胞嘧啶（5 hmC）的蛋白质，5-羟甲基胞嘧啶是由泰特蛋白产生的表观遗传修饰。我们的工作表明，它含有一个催化三联体，可能具有自肽酶活性。据报道，该活性位点还通过其保守的半胱氨酸在脱碱基位点与DNA共价交联。在与Anjana Rao博士实验室的合作中，我们发现Hmces缺陷小鼠显示正常的造血功能，而5 hmC没有整体改变。在B细胞中的类别转换重组（CSR）期间，HMCES通过微同源介导的交替末端连接（Alt-EJ）途径特异性地实现DNA双链断裂修复，HMCES缺陷导致CSR的显著缺陷。HMCES通过其SOS反应相关肽酶结构域（SRAPd）介导Alt-EJ，该功能需要DNA结合，但不依赖于其自肽酶和DNA交联活性。我们发现，HMCES被募集到免疫球蛋白基因座的开关区域，并为HMCES与CSR期间Alt-EJ产生的长DNA突出端的相互作用提供了潜在的结构基础。我们的研究为HMCES作为起源于细菌的真核DNA修复的新参与者提供了强有力的证据。 新型冠状病毒（SARS-CoV-2）是人类突发严重呼吸道疾病（COVID-19）的病原体，已导致全球健康危机。通过基因组、序列、结构和进化分析，我们鉴定了几个在SARS-CoV-2中快速进化的蛋白，这些蛋白在发病机制中具有潜在的作用。除了众所周知的刺突蛋白外，这些包括预测加工NAD+的宏结构域三联体，并且表明α-和β-CoV具有几个新的免疫球蛋白（IG）结构域蛋白家族，包括来自SARS相关冠状病毒的ORF 8和ORF 7a以及来自某些α-CoV的两个蛋白质组。其中，ORF 8的特点是进化迅速，在其预测的配体结合沟中具有独特的插入片段和SARS-CoV-2基因组中的高变位置。我们还发现了许多IG蛋白从几个后生动物病毒，这是不同的序列和结构，但共享的架构，冠状病毒IG结构域蛋白。因此，我们认为IG结构域蛋白的部署是病毒广泛使用的策略，并且SARS-CoV-2 ORF 8是一个潜在的致病因子，其快速进化以对抗免疫应答并促进宿主之间的传播。 社会细胞聚集或多细胞组织在单个细胞感染时增加了通过系统传播感染的风险。后生动物以外的进化反应的一般性仍然不清楚。我们发现了几个主题统一，显着的生物冲突系统主要存在于多细胞原核生物。这些联合收割机结合了利用NTR分子伴侣（MoxR-vWA偶联）、GTP酶和蛋白水解级联的阈值机制与高变效应子，其通过使用逆转录酶依赖性多样性生成系统或通过从各种细胞子系统获取多样性蛋白模块（通常为无活性形式）的系统而变化。有利的证据表明，它们对入侵实体（如病毒）的部署，通过物理遏制、显性-负性相互作用或细胞凋亡来限制它们在多细胞/社会环境中的传播。基于这些发现，我们认为，在多细胞生物的多次出现期间，在感测和限制感染方面，存在类似的操作“语法”和共享的蛋白质结构域。

The origin of eukaryotes was marked by the emergence of several novel subcellular systems. One such is the calcium (Ca2+)-stores system of the endoplasmic reticulum, which profoundly influences diverse aspects of cellular function including signal transduction, motility, division, and biomineralization. We used comparative genomics and sensitive sequence and structure analyses to investigate the evolution of this system. Our findings reconstruct the core form of the Ca2+-stores system in the last eukaryotic common ancestor as having at least 15 proteins that constituted a basic system for facilitating both Ca2+ flux across endomembranes and Ca2+-dependent signaling. We showed that the key EF-hand Ca2+-binding components had their origins in a likely bacterial symbiont other than the mitochondrial progenitor, whereas the protein phosphatase subunit of the ancestral calcineurin complex was likely inherited from the asgardarchaeal progenitor of the stem eukaryote. This further points to the potential origin of the eukaryotes in a Ca2+-rich biomineralized environment such as stromatolites. We further show that throughout eukaryotic evolution there were several acquisitions from bacteria of key components of the Ca2+-stores system, even though no prokaryotic lineage possesses a comparable system. Further, using quantitative measures derived from comparative genomics we show that there were several rounds of lineage-specific gene expansions, innovations of novel gene families, and gene losses correlated with biological innovation such as the biomineralized molluscan shells, coccolithophores, and animal motility. The burst of innovation of new genes in animals included the wolframin protein associated with Wolfram syndrome in humans. We showed for the first time that it contains previously unidentified Sel1, EF-hand, and OB-fold domains, which might have key roles in its biochemistry. HMCES (5hmC binding, embryonic stem cell-specific-protein) was originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins. Our work showed that it contains a catalytic triad that is likely to possess autopeptidase activity. This active site was also reported to covalently crosslink to DNA at abasic sites via its conserved cysteine. In a collaboration with Dr. Anjana Rao's lab we showed that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We showed that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provided strong evidence for for HMCES as a novel player in eukaryotic DNA repair with an origin in bacteria. The novel coronavirus (SARS-CoV-2) is the causative agent of an emergent severe respiratory disease (COVID-19) in humans that has resulted in a global health crisis. By using genomic, sequence, structural and evolutionary analysis, we identified several rapidly evolving proteins in SARS-CoV-2 with potential roles in pathogenesis. In addition to the well-known spike protein, these include the triad of Macro domains predicted to process NAD+ an showed that Alpha- and Beta-CoVs possess several novel families of immunoglobulin (Ig) domain proteins, including ORF8 and ORF7a from SARS-related coronaviruses and two protein groups from certain Alpha-CoVs. Among them, ORF8 is distinguished in being rapidly evolving, possessing a unique insert and a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncovered many Ig proteins from several metazoan viruses, which are distinct in sequence and structure but share an architecture comparable to that of CoV Ig domain proteins. Hence, we propose that deployment of Ig domain proteins is a widely-used strategy by viruses, and SARS-CoV-2 ORF8 is a potential pathogenicity factor which evolves rapidly to counter the immune response and facilitate the transmission between hosts. Social cellular aggregation or multicellular organization pose increased risk of transmission of infections through the system upon infection of a single cell. The generality of the evolutionary responses to this outside of Metazoa remains unclear. We discovered several thematically unified, remarkable biological conflict systems preponderantly present in multicellular prokaryotes. These combine thresholding mechanisms utilizing NTPase chaperones (the MoxR-vWA couple), GTPases and proteolytic cascades with hypervariable effectors, which vary either by using a reverse transcriptase-dependent diversity-generating system or through a system of acquisition of diverse protein modules, typically in inactive form, from various cellular subsystems. Conciliant lines of evidence indicate their deployment against invasive entities, like viruses, to limit their spread in multicellular/social contexts via physical containment, dominant-negative interactions or apoptosis. Base on these findings we argue for both a similar operational 'grammar' and shared protein domains in the sensing and limiting of infections during the multiple emergences of multicellularity.