Evolutionary Analysis and Comparative Genomics of Protein Superfamilies

真核生物的起源标志着几个新的亚细胞系统的出现。其中之一是内质网的钙储存系统，它深刻地影响着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和敏感的序列和结构分析来研究这个系统的进化。我们的发现重建了真核生物最后一个共同祖先的钙储存系统的核心形式，即至少有15个蛋白质，构成了促进跨内膜钙流动和钙依赖信号传递的基本系统。我们表明，EF-Hand钙结合的关键成分可能起源于一个可能的细菌共生体，而不是线粒体前体，而祖先钙调神经磷酸酶复合体的蛋白磷酸酶亚单位可能遗传自茎真核生物的子囊前体。这进一步指出了真核生物在富钙生物矿化环境(如叠层石)中的潜在起源。我们进一步表明，在整个真核进化过程中，有几次从细菌那里获得了钙离子储存系统的关键成分，尽管没有一个原核谱系拥有可比的系统。此外，使用来自比较基因组学的定量方法，我们发现有几轮谱系特异的基因扩张，新基因家族的创新，以及与生物创新相关的基因丢失，如生物矿化的软体贝壳、球虫基团和动物运动性。动物新基因的突破性创新包括与人类Wolfram综合征相关的狼帧蛋白。我们首次证明它含有以前未确定的Sel1、EF-Hand和OB-折叠结构域，这些结构域可能在其生物化学中发挥关键作用。 HMCES(5hmC结合，胚胎干细胞特异性蛋白)最初被鉴定为能够与5-羟甲基胞嘧啶(5hmC)结合的蛋白质，5-羟甲基胞嘧啶(5hmC)是由Tet蛋白产生的表观遗传修饰。我们的工作表明，它含有一种催化三联体，很可能具有自体多肽酶活性。据报道，该活性部位还通过其保守的半胱氨酸与基本部位的DNA发生共价交联。在与Anjana Rao博士的实验室的合作中，我们表明Hmcs缺陷小鼠表现出正常的造血功能，5hmC没有整体变化。在B细胞的类开关重组(CSR)过程中，HMCES通过微同源介导的交替末端连接(Alt-EJ)途径特异性地使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，这一功能需要DNA结合，但不依赖于其自体多肽酶和DNA交联活性。我们发现HMCES被招募来切换免疫球蛋白基因座的区域，并为HMCES与Alt-EJ在CSR过程中产生的长DNA悬垂相互作用提供了潜在的结构基础。我们的研究为HMCES作为一种新的起源于细菌的真核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)是人类一种紧急严重呼吸道疾病(新冠肺炎)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SARS-CoV-2中几个快速进化的蛋白，它们可能在致病机制中发挥作用。除了已知的Spike蛋白外，这些蛋白还包括被预测为处理NAD+AN的三个宏结构域的三联体。结果表明，Alpha和Beta冠状病毒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ORF8和ORF7a以及来自某些Alpha冠状病毒的两个蛋白质组。其中，ORF8基因进化迅速，在SARS-CoV-2基因组中拥有唯一的插入片段，并在其预测的配体结合槽中具有高度可变的位置。我们还从几种后生动物病毒中发现了许多Ig蛋白，它们在序列和结构上有所不同，但具有与冠状病毒Ig结构域蛋白相似的结构。因此，我们认为部署Ig结构域蛋白是病毒广泛使用的策略，而SARS-CoV-2 ORF8是一个潜在的致病因子，它快速进化以对抗免疫反应并促进宿主之间的传播。 社会细胞聚集或多细胞组织在感染单个细胞时会增加通过系统传播感染的风险。后生动物以外对此的进化反应的普遍性尚不清楚。我们发现了几个主题统一的、显著的生物冲突系统，它们主要存在于多细胞原核生物中。这些机制结合了利用NTPase伴侣(MoxR-VWA偶联)、GTP酶和具有高变量效应器的蛋白水解级联的阈值机制，这些效应器要么通过使用依赖于逆转录酶的多样性产生系统改变，要么通过从各种细胞子系统获取不同的蛋白质模块的系统改变，通常是以不活跃的形式。温和的证据表明，它们针对病毒等侵入性实体进行部署，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，我们认为在多细胞的多重出现期间，在感知和限制感染方面，我们既有类似的操作‘语法’，也有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.