BACKGROUND & AIMS: To delineate the cis-acting elements of the proximal promoter responsible for cyclic AMP (cAMP)-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4-fold when the reporter plasmid contained the promoter region (-582 to + 16). Mutation or deletion of the cAMP response element (CRE) diminished (1.7-fold) but did not abolish cAMP-induced transcription, demonstrating that the (-582 to -145) region containing the CRE and the region (-145 to -38) containing a Pit-1 (pituitary-specific trans-acting factor) site were both necessary for cAMP maximal induction. To study the molecular events mediating the cAMP induction, DNase I footprinting and electromobility shift assays (EMSAs) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, showing that the CRE-binding protein (CREB) interacts with the CRE and that tissue-specific factors, distinct from Pit-1, specifically bind the renin Pit-1 motif. Taken together, these results demonstrate that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors, different from Pit-1, that interact with the Pit-1 response DNA elements.

背景与目标： 为了描绘负责环AMP (cAMP) 诱导的人肾素基因转录的近端启动子的顺式作用元件，将人肾素基因的5 '侧翼区域融合到荧光素酶报告基因上，并转染到绒毛膜细胞中。当报道质粒包含启动子区域 (-582至 + 16) 时，福司可林处理诱导荧光素酶的表达2.4倍。cAMP反应元件 (CRE) 的突变或缺失减少 (1.7倍)，但没有消除cAMP诱导的转录，证明含有CRE的 (-582至-145) 区域和含有Pit-1 (垂体特异性反式作用因子) 位点的区域 (-145至-38) 对于cAMP最大诱导都是必需的。为了研究介导cAMP诱导的分子事件，使用产生肾素的绒毛膜细胞和肾皮质细胞核提取物进行了DNase I足迹和电迁移率位移测定 (EMSAs)，表明CRE结合蛋白 (CREB) 与CRE相互作用，并且组织特异性因素，与Pit-1不同，特异性结合肾素Pit-1图案。总之，这些结果表明，人类肾素基因的cAMP反应可能涉及CREB结合CRE和与Pit-1反应DNA元件相互作用的不同于Pit-1的组织特异性因子。

BACKGROUND & AIMS: :In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates. Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs. The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.

背景与目标： : 为了鉴定强的跨膜螺旋堆积基序，我们根据糖蛋白a的右旋二聚化基序从随机序列文库中选择了表现出高亲和力同型寡聚的跨膜结构域。使用TOXCAT系统分离序列，该系统测量大肠杆菌内膜中的跨膜螺旋-螺旋缔合。强选择被应用于大范围的序列 (大约10(7) 个可能性)，并导致识别介导高亲和力螺旋-螺旋缔合的序列模式。分离出的最常见的基序GxxxG发生在超过80% 的分离物中。其他相关性表明，侧翼残基与GxxxG基序协同作用，并且在界面处保持大小互补性，这与确定的序列模式代表堆积基序的想法一致。通过对SwissProt序列数据库中的跨膜结构域的分析，对相似序列模式的趋同鉴定表明，这些堆积基序经常用于天然存在的螺旋膜蛋白中。

BACKGROUND & AIMS: :Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.

背景与目标： : 双子座病毒es编码一种复制起始蛋白Rep，该蛋白以序列特异性方式与作为病毒特异性复制必需元素的迭代DNA基序 (iterons) 结合。通过使用超过100个双生病毒的迭代作为启发式设备，我们已经确定了一个长度为8到10个残基的Rep子域，其一级结构在具有不同迭代的病毒中有所不同，但在具有相同迭代的病毒中是相似的，无论它们在宿主范围，昆虫载体，地理起源或基因组结构。对该iteron相关结构域 (IRD) 的仔细分析显示，特定Rep残基与其同源iteron的定义核苷酸之间存在一致的相关性，从而提供了有关决定Rep对特定DNA序列偏好的分子代码的重要见解。提出了一种潜在代表联系人模型。鉴定出的IRD与滚动圈 (RC) 复制蛋白超家族的保守基序特征相邻，二级结构预测表明，这些Rep子域共同形成了具有 β-折叠的新型DNA结合域的核心作为识别子域，在纳米病毒，圆环病毒，微病毒以及真细菌，古细菌和红藻的各种ssDNA质粒的复制蛋白中显然是保守的。讨论了这些发现的进化意义。

BACKGROUND & AIMS: :Due to limited available therapeutic options, developing new lead compounds against hepatitis C virus is an urgent need. Human La protein stimulates hepatitis C virus translation through interaction with the hepatitis C viral RNA. A cyclic peptide mimicking the β-turn of the human La protein that interacts with the viral RNA was synthesized. It inhibits hepatitis C viral RNA translation significantly better than the corresponding linear peptide at longer post-treatment times. The cyclic peptide also inhibited replication as measured by replicon RNA levels using real time RT-PCR. The cyclic peptide emerges as a promising lead compound against hepatitis C.

背景与目标： : 由于可用的治疗选择有限，迫切需要开发新的抗丙型肝炎病毒的领先化合物。人La蛋白通过与丙型肝炎病毒RNA相互作用刺激丙型肝炎病毒翻译。合成了模拟与病毒RNA相互作用的人La蛋白的 β-转的环肽。在较长的治疗后时间，它对丙型肝炎病毒RNA翻译的抑制作用明显优于相应的线性肽。环肽还抑制复制，如使用实时rt-pcr通过复制子RNA水平测量的那样。环肽作为抗丙型肝炎的有前途的铅化合物出现。

BACKGROUND & AIMS: :Non-structural protein (nsp) 1 of PRRS virus is a viral antagonist for type I interferons (IFNs), and in cells expressing nsp1, CREB-binding protein (CBP) is degraded. nsp1 is auto-processed into nsp1α and nsp1β subunits and in the present study we show that the nsp1α subunit was responsible for CBP degradation. The nsp1α subunit contains three distinct functional motifs; a papain-like cysteine protease α (PCPα) motif, an N-terminal zinc finger motif (ZF1), and a newly reported C-terminal zinc finger motif (ZF2). To study the structure function of nsp1α and its IFN antagonism, these motifs were individually mutated and the mutants were examined for their IFN suppression ability. The mutations that destroyed the PCPα activities (C76S, H146Y, and C76S/H146Y) did not affect the IFN suppressive activity of nsp1α, indicating that the cysteine protease activity did not participate in IFN suppression. The mutations of C70S, C76S, H146Y, and/or M180I which coordinated the ZF2 motif also did not alter IFN suppression. However, the mutations of C8S, C10S, C25S, and/or C28S for the ZF1 motif impaired the IFN antagonism of nsp1α, demonstrating that ZF1 was the essential element of nsp1α for IFN suppression. Wild-type nsp1α localized in the both nucleus and cytoplasm, but the ZF1 mutants that lost the IFN suppressive activity did not localize in the nucleus and remained in the cytoplasm. Consistent with their cytoplasmic distribution, CBP was not degraded by these mutants. Our results indicate that the ZF1 motif of nsp1α plays an important role for IFN regulation and further demonstrate that the CBP degradation is likely the key mechanism for IFN suppression mediated by the nsp1α subunit protein of PRRS virus.

背景与目标： : PRRS病毒的非结构蛋白 (nsp) 1是I型干扰素 (ifn) 的病毒拮抗剂，在表达nsp1的细胞中，CREB结合蛋白 (CBP) 被降解。nsp1被自动加工成nsp1α 和nsp1β 亚基，在本研究中，我们表明nsp1α 亚基是CBP降解的原因。nsp1α 亚基包含三个不同的功能基序; 木瓜蛋白酶样半胱氨酸蛋白酶 α (pcp α) 基序，N端锌指基序 (ZF1) 和新报道的C端锌指基序 (ZF2)。为了研究nsp1α 的结构功能及其IFN拮抗作用，对这些基序进行了单独突变，并检查了突变体的IFN抑制能力。破坏pcp α 活性的突变 (C76S，H146Y和C76S/H146Y) 不影响nsp1α 的IFN抑制活性，表明半胱氨酸蛋白酶活性不参与IFN抑制。协调ZF2基序的C70S，C76S，H146Y和/或M180I的突变也不会改变IFN抑制。然而，ZF1基序的C8S，C10S，C25S和/或C28S突变损害了nsp1α 的IFN拮抗作用，表明ZF1是nsp1α 抑制IFN的基本要素。野生型nsp1α 位于细胞核和细胞质中，但是失去IFN抑制活性的ZF1突变体未定位在细胞核中，而保留在细胞质中。与它们的细胞质分布一致，这些突变体不会降解CBP。我们的结果表明，nsp1α 的ZF1基序在IFN调节中起着重要作用，并进一步证明CBP降解可能是PRRS病毒nsp1α 亚基蛋白介导的IFN抑制的关键机制。

BACKGROUND & AIMS: :Endocytosis of the amyloid precursor protein (APP) is critical for generation of β-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.

背景与目标： : 淀粉样前体蛋白 (APP) 的内吞作用对于 β-淀粉样蛋白的生成至关重要，在阿尔茨海默氏病中聚集。取决于细胞内NPTY基序的APP内吞作用已得到很好的研究，而YTSI (也称为低音) 基序的参与仍存在争议。在这里，我们显示缺少YTSI基序 (Δ YTSI) 的APP显示出对早期内体的定位降低，内化率降低，类似于APP Δ npty。此外，我们显示YTSI结合蛋白PAT1a与Rab5激活剂RME-6相互作用，如几个独立的试验所示。有趣的是，RME-6的敲低降低了APP内吞作用，而过表达却增加了。同样，APP Δ npty内吞作用受PAT1a和RME-6过表达的影响，而APP Δ ytsi内化保持不变。此外，我们可以表明，在击倒PAT1a时，RME-6介导的APP内吞作用的增加可以减少。总之，我们的数据将RME-6识别为APP内吞作用的新参与者，涉及YTSI结合蛋白PAT1a。

BACKGROUND & AIMS: :Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.

背景与目标： : 选择性自噬是自噬在多细胞生物中发挥的许多重要生理作用的基础，但是对货物选择所涉及的机制知之甚少。在这里，我们描述了一种分子机制，该机制可以靶向常规内体进行自噬降解。我们显示，人跨膜蛋白TMEM59在其细胞内结构域中包含最小19个氨基酸的肽，可促进LC3标记和溶酶体靶向其自身的内体区室。有趣的是，该肽定义了一种新型的蛋白质基序，该基序介导与ATG16L1的WD重复结构域的相互作用，从而为该活性提供了机械基础。该基序在其他分子中以相同的ATG16L1-binding能力表示，表明更普遍的相关性。我们认为，该基序可能在靶向特定的膜区室进行自噬降解中起重要作用，因此，它可能有助于寻找通过参与ATG16L1促进选择性自噬的衔接子蛋白。内源性TMEM59与ATG16L1相互作用并介导自噬以响应金黄色葡萄球菌感染。

BACKGROUND & AIMS: :Major histocompatibility complex II (MHC II) molecules play a vital role in the onset and control of cellular immunity. In a highly selective process, MHC II presents peptides derived from exogenous antigens on the surface of antigen-presenting cells for T cell scrutiny. Understanding the rules defining this presentation holds critical insights into the regulation and potential manipulation of the cellular immune system. Here, we apply the NNAlign_MA machine learning framework to analyze and integrate large-scale eluted MHC II ligand mass spectrometry (MS) data sets to advance prediction of CD4+ epitopes. NNAlign_MA allows integration of mixed data types, handling ligands with multiple potential allele annotations, encoding of ligand context, leveraging information between data sets, and has pan-specific power allowing accurate predictions outside the set of molecules included in the training data. Applying this framework, we identified accurate binding motifs of more than 50 MHC class II molecules described by MS data, particularly expanding coverage for DP and DQ beyond that obtained using current MS motif deconvolution techniques. Furthermore, in large-scale benchmarking, the final model termed NetMHCIIpan-4.0 demonstrated improved performance beyond current state-of-the-art predictors for ligand and CD4+ T cell epitope prediction. These results suggest that NNAlign_MA and NetMHCIIpan-4.0 are powerful tools for analysis of immunopeptidome MS data, prediction of T cell epitopes, and development of personalized immunotherapies.

背景与目标： : 主要组织相容性复合体II (MHC II) 分子在细胞免疫的发作和控制中起着至关重要的作用。在高度选择性的过程中，MHC II在抗原呈递细胞表面呈递源自外源抗原的肽，用于T细胞检查。了解定义此演示文稿的规则具有对细胞免疫系统的调节和潜在操纵的重要见解。在这里，我们应用NNAlign_MA机器学习框架来分析和整合大规模洗脱的MHC II配体质谱 (MS) 数据集，以推进CD4表位的预测。NNAlign_MA允许混合数据类型的集成，处理具有多个潜在等位基因注释的配体，对配体上下文进行编码，利用数据集之间的信息，并且具有泛特异性的能力，允许在训练数据中包含的分子集之外进行准确的预测。应用此框架，我们确定了MS data描述的50多个MHC II类分子的准确结合基序，尤其是将DP和DQ的覆盖范围扩大到使用当前MS motif反卷积技术获得的范围之外。此外，在大规模基准测试中，被称为NetMHCIIpan-4.0的最终模型展示了超越当前配体和CD4 + T细胞表位预测的最新预测指标的改进性能。这些结果表明，NNAlign_MA和NetMHCIIpan-4.0是分析免疫肽MS数据，预测T细胞表位和开发个性化免疫疗法的强大工具。

BACKGROUND & AIMS: :Objective: Cancer stem cell is one of the important causes of tumorigenesis as well as a drug target in the treatment of malignant tumor. However, at present, there is no immune vaccine targeting these cells. Octamer-binding transcription factor 4 (OCT4), a marker of embryonic stem cells and germ cells, often highly expresses in the early stages of tumorigenesis and is therefore a good candidate for cancer vaccine development. Methods: To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked three different OCT4 epitope antigens to a carrier protein, keyhole limpet hemocyanin (KLH), combined with Toll-like receptor 9 agonist (TLR9). Results: Immunization with OCT4-3 + TLR9 produced the strongest immune response in mice. In prevention assays, significant tumor growth inhibition was achieved in BABL/c mice treated with OCT4-3 + TLR9 (P < 0.01). Importantly, the results showed that cytotoxic T lymphocyte activity and the inhibition of tumor growth were enhanced in mice immunized with OCT4-3 combined with TLR9. Meanwhile, multiple cytokines [such as interferon (IFN)-γ (P < 0.05), interleukin (IL)-12 (P < 0.05), IL-2 (P < 0.01), and IL-6 (P < 0.05)] promoting cellular immune responses were shown to be greatly enhanced in mice immunized with OCT4-3 + TLR9. Moreover, we considered safety considerations in terms of the composition of the vaccines to help facilitate the development of effective next-generation vaccines. Conclusions: Collectively, these experiments demonstrated that combination therapy with TLR9 agonist induced a tumor-specific adaptive immune response, leading to the suppression of primary tumor growth in testis embryonic carcinoma.

背景与目标： 目的: 肿瘤干细胞是肿瘤发生的重要原因之一，也是治疗恶性肿瘤的药物靶点。但是，目前还没有针对这些细胞的免疫疫苗。八聚体结合转录因子4 (OCT4) 是胚胎干细胞和生殖细胞的标志物，通常在肿瘤发生的早期阶段高度表达，因此是癌症疫苗开发的良好候选者。方法: 为了确定最佳的载体和佐剂组合，我们化学合成了三种不同的OCT4表位抗原，并将其与载体蛋白匙孔血蓝蛋白 (KLH) 和Toll样受体9激动剂 (TLR9) 结合。结果: OCT4-3 + TLR9预防接种小鼠免疫反应最强。在预防试验中，在用OCT4-3 + TLR9处理的BABL/c小鼠中实现了显著的肿瘤生长抑制 (P <0.01)。重要的是，结果表明，OCT4-3联合tlr9免疫小鼠的细胞毒性T淋巴细胞活性和对肿瘤生长的抑制作用增强。同时，多种细胞因子 [干扰素 (IFN)-γ (P <0.05)，白细胞介素 (IL)-12 (P <0.05)，IL-2 (P <0.01)，并且IL-6 (P < 0.05)] 促进细胞免疫应答在用OCT4-3 + tlr9免疫的小鼠中显示出显著增强。此外，我们考虑了疫苗组成方面的安全性考虑，以帮助促进有效的下一代疫苗的开发。结论: 总的来说，这些实验表明，与TLR9激动剂联合治疗可诱导肿瘤特异性适应性免疫反应，从而抑制睾丸胚胎癌的原发性肿瘤生长。

BACKGROUND & AIMS: :Protein export in intra-erythrocytic Plasmodium parasites is of considerable interest in the malaria field because the process is inextricably linked to virulence and survival mechanisms in the human host. Despite many and varied functions, a common link between many exported proteins is their actual mode of export. Most exported proteins must traverse two membranes to their destination in the infected erythrocyte cytosol, the parasite plasma membrane and surrounding parasitophorous vacuole membrane (PVM). In recent years, several studies have shone light on the common molecular mechanism by which the major class of exported proteins, the so-called PEXEL/HTS motif-containing proteins, are translocated across these membranes. Roles for parasite-specific molecular processes in two distinct sites, the endoplasmic reticulum and the PVM have been revealed.

背景与目标： : 红细胞内疟原虫寄生虫中的蛋白质输出在疟疾领域引起了极大的兴趣，因为该过程与人类宿主的毒力和生存机制密不可分。尽管功能多种多样，但许多出口蛋白质之间的共同联系是它们的实际出口方式。大多数出口的蛋白质必须穿过两个膜到达被感染的红细胞胞质溶胶，寄生虫质膜和周围的寄生虫液泡膜 (PVM) 中的目的地。近年来，一些研究揭示了常见的分子机制，通过这种机制，主要的出口蛋白，即所谓的含PEXEL/HTS基序的蛋白质，在这些膜上易位。已揭示了寄生虫特异性分子过程在两个不同部位的作用，即内质网和PVM。

BACKGROUND & AIMS: :Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.

背景与目标： : 轴突再生是脊髓损伤后功能恢复的必要步骤。众所周知，AP-1转录因子c-6月在指导背根神经节 (DRG) 神经元对外周轴突切断术的转录反应中发挥重要作用，从而成功实现轴突再生。在这里，我们在坐骨神经挤压或假手术后对小鼠DRG神经元进行了ChIPseq 6月，以测量6月对外周轴切开术的DNA结合变化。我们发现，6月在DNA结合中的大多数损伤反应性变化都发生在假定的增强子元件上，而不是在转录起始位点附近。我们还使用了一系列单多肽链串联转录因子来测试不同的含6月二聚体对DRG，皮质和海马神经元的神经突生长的影响。这些实验表明，由6月和Atf3组成的二聚体可促进大鼠CNS神经元以及小鼠DRG神经元的神经突生长。我们的工作为6月在轴突再生中的作用机制提供了新的见解。

BACKGROUND & AIMS: :RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.

背景与目标： : RNA是开发功能化学探针或小分子疗法的极其重要的靶标。氨基糖苷类是研究最充分的一类靶向RNA的小分子。然而，在生物系统中可能被这些化合物结合的细菌rRNA A位点之外的RNA基序在很大程度上是未知的。如果已知此类信息，则可以利用氨基糖苷类药物靶向其他rna，此外，还可以为这些临床使用的药物的潜在旁观者靶标提供宝贵的见解。我们利用二维组合筛选 (2DCS) (一种库对库筛选方法) 来选择在3 × 3核苷酸内环文库和6核苷酸发夹文库中显示的基序，这些基序以高亲和力和选择性结合六个氨基糖苷衍生物。然后通过测序 (ststarts) 使用结构-活性关系分析所选的RNA基序，这是一种统计方法，定义了结合小分子的特权RNA基序空间。开始允许在亲和力和选择性方面轻松注释所选RNA基序-氨基糖苷类相互作用。由2dc选择的相互作用通常具有纳摩尔亲和力，这比氨基糖苷类与其治疗靶标细菌rRNA a位点的模拟物结合的亲和力更高。

BACKGROUND & AIMS: :In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

背景与目标： : 在对抗抗菌素耐药性的斗争中，细菌DNA滑动钳 β-钳是抑制DNA复制和跨病变合成的有前途的药物靶标。Β-clamp及其真核同源物PCNA共享一个C末端疏水口袋，所有DNA聚合酶都在其中结合。在这里，我们报告了含有PCNA相互作用基序APIM (APIM-肽) 的细胞穿透肽在体外和体内抑制细菌生长在小鼠细菌皮肤感染模型中的低浓度。表面等离子体共振分析和计算机建模表明，APIM与 β-clamp上的疏水口袋结合，因此，我们发现APIM-肽抑制细菌DNA复制。有趣的是，在亚致死浓度下，APIM肽具有抗诱变活性，并且该活性在SOS诱导后增加。我们的结果表明，尽管 β-clamp和PCNA之间的序列同源性适中，但DNA clamp中类似的聚合酶结合袋的存在允许真核结合基序APIM与细菌 β-clamp结合。重要的是，由于APIM肽同时具有抗诱变和生长抑制特性，因此它们可能具有与其他抗生素联合使用以及作为单一药物的临床潜力。

BACKGROUND & AIMS: :The protein kinase A-anchoring proteins (AKAPs) are defined by their ability to scaffold protein kinase A to specific subcellular compartments. Each of the AKAP family members utilizes unique targeting domains specific for a particular subcellular compartment. AKAP350 is a multiply spliced AKAP family member localized to the centrosome and the Golgi apparatus. Three splicing events in the carboxyl terminus of AKAP350 generate the AKAP350A, AKAP350B, and AKAP350C proteins. A monoclonal antibody recognizing all three splice variants as well as a polyclonal antibody specific for AKAP350A demonstrated both centrosomal and Golgi apparatus staining in paraformaldehyde-fixed HCA-7 cells. Golgi apparatus-associated AKAP350A staining was dispersed following brefeldin A treatment. Using GFP chimeric constructs of the carboxyl-terminal regions of AKAP350A, a Golgi apparatus targeting domain was identified between amino acids 3259 and 3307 of AKAP350A. This domain was functionally distinguishable from the recently described centrosomal targeting domain (PACT domain, amino acids 3308-3324) located adjacent to the Golgi targeting domain. These data definitively establish the specific association of AKAP350A with the Golgi apparatus in HCA-7 cells.

背景与目标： : 蛋白激酶A锚定蛋白 (akap) 的定义是通过其将蛋白激酶A支架到特定亚细胞区室的能力。每个AKAP家族成员都使用特定于特定亚细胞区室的独特靶向域。AKAP350是一个多重拼接的AKAP家族成员，定位于中心体和高尔基体。AKAP350羧基末端的三个剪接事件生成AKAP350A，AKAP350B和AKAP350C蛋白。识别所有三个剪接变体的单克隆抗体以及对AKAP350A具有特异性的多克隆抗体在多聚甲醛固定的HCA-7细胞中证实了中心体和高尔基体染色。brefeldin A处理后，将高尔基体相关的AKAP350A染色分散。使用AKAP350A的羧基末端区域的GFP嵌合构建体，在AKAP350A的氨基酸3259和3307之间鉴定了高尔基体靶向结构域。该结构域在功能上可与最近描述的邻近高尔基体靶向结构域的中心体靶向结构域 (PACT结构域，氨基酸3308-3324) 区分开来。这些数据确定了AKAP350A与HCA-7细胞中高尔基体的特异性关联。

BACKGROUND & AIMS: :COMP (cartilage oligomeric matrix protein) is a member of the thrombospondin family and forms homopentamers as well as mixed heterooligomers with its closely related family member TSP-4. COMP is long known to bind to collagens and to influence collagen fibril formation. Recent work indicates that already intracellular interaction with collagen is important for collagen secretion. However, the exact binding site of COMP on the collagen triple helix has not been described up to now. In this study we have identified a GXKGHR motif on the collagen II helix to bind to COMP, using a recombinantly expressed collagen II peptide library. This binding sequence is conserved throughout evolution and we demonstrate that TSP-4 binds to the same sequence. The identified binding motif overlaps with the recognition sites of many other collagen-binding partners (e.g. PEDF, Heparin) and also spans the lysine residues, which form collagen cross-links. COMP might thereby protect collagen helices from premature modification and cross-linking. Interestingly, this motif is only found in classical fibrillar collagens, although COMP is known to also bind other types. This might indicate that COMP has a unique interface for fibrillar collagens, thus making it an interesting target for the development of antifibrotic drugs.

背景与目标： : COMP (软骨寡聚基质蛋白) 是血小板反应蛋白家族的成员，并与其密切相关的家族成员TSP-4形成同型聚体以及混合的杂低聚物。COMP长期以来一直与胶原蛋白结合并影响胶原蛋白原纤维的形成。最近的工作表明，与胶原蛋白的细胞内相互作用对于胶原蛋白的分泌很重要。然而，到目前为止，尚未描述COMP在胶原蛋白三螺旋上的确切结合位点。在这项研究中，我们使用重组表达的胶原蛋白II肽库鉴定了胶原蛋白II螺旋上的GXKGHR基序与COMP结合。该结合序列在整个进化过程中是保守的，我们证明TSP-4与同一序列结合。鉴定出的结合基序与许多其他胶原蛋白结合伴侣 (例如PEDF，肝素) 的识别位点重叠，并且还跨越赖氨酸残基，这些赖氨酸残基形成胶原蛋白交联。COMP可能因此保护胶原蛋白螺旋免于过早修饰和交联。有趣的是，该基序仅在经典原纤维胶原蛋白中发现，尽管已知COMP也结合其他类型。这可能表明COMP具有用于原纤维胶原蛋白的独特界面，因此使其成为开发抗纤维化药物的有趣靶标。