BACKGROUND & AIMS: :In the present study we investigated the relation between cyclophosphamide and methotrexate toxicity and the presence of HLA- DR B1 alleles in rheumatoid arthritis patients. Seventy-eight such patients (67 women and 11 men) were observed for 12 months. Eighteen were treated with intravenous cyclophosphamide, 28 with oral methotrexate, and 32 with intramuscular gold salts. The prevalence of this shared motif was higher in the study population than in the healthy controls. However, detailed observations did not demonstrate a relation between particular genotype and drug intolerance. Based on the obtained findings we concluded that HLA-DR B1 typing cannot affect cyclophosphamide or methotrexate tolerance in rheumatoid arthritis patients. However, taking into account the relatively small number of patients expressing single genotype, further studies are recommended.

背景与目标： : 在本研究中，我们调查了类风湿关节炎患者中环磷酰胺和甲氨蝶呤毒性与hla-dr B1等位基因的存在之间的关系。观察了78名此类患者 (67名女性和11名男性) 12个月。静脉注射环磷酰胺治疗18例，口服甲氨蝶呤治疗28例，肌内注射金盐治疗32例。研究人群中这种共有基序的患病率高于健康对照组。然而，详细的观察结果并未证明特定基因型与药物不耐受之间的关系。根据获得的发现，我们得出结论，hla-dr B1分型不会影响类风湿关节炎患者的环磷酰胺或甲氨蝶呤耐受性。但是，考虑到表达单一基因型的患者数量相对较少，建议进一步研究。

BACKGROUND & AIMS: :High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

背景与目标： : 高通量TCR测序允许询问人类TCR库，有可能将TCR序列与抗原靶标连接起来。与高度多态的MHC蛋白不同，单型Ag呈递分子 (例如MR1，CD1d和CD1b) 向具有物种范围的TCR基序的T细胞呈递Ags。CD1b四聚体研究和对27个已发表的CD1b-restricted TCR的调查表明，人类的TCR基序由TCR β 链可变基因4-1 (TRBV4-1) 区域定义。出乎意料的是，无论所携带脂质的化学类别如何，TRBV4-1都参与了CD1b的识别。两个CD1b-specific TRBV4-1 TCR的晶体结构表明，TRBV4-1-encoded序列的CDR1和CDR3区域中的种系编码残基相互作用并巩固TCR的表面。突变研究确定了TRBV4-1中的关键带正电残基和与CD1c共享的CD1b中的关键带负电残基，这也被TRBV4-1 tcr识别。这些数据表明，一个TCR V区可以介导两个相关的单态Ag呈递分子的识别机制，这些分子不依赖于定义的脂质Ag。

BACKGROUND & AIMS: :The UvrB subunit is a central component of the UvrABC incision complex and plays a pivotal role in damage recognition, strand excision and repair synthesis. A conserved structural motif (the SxSx motif) present in UvrB is analogous to a similar motif (TxGx) in the helicases of superfamily 2, whose function is not fully understood. To elucidate the significance of the SxSx (Ser143-Val144-Ser145-Cys146) motif in Mycobacterium tuberculosis UvrB (MtUvrB), different variants of MtUvrB subunit were constructed and characterized. The SxSx motif indeed was found to be essential for MtUvrB function: while Ser143 and Cys146 residues within this motif were crucial for MtUvrB function, Ser145 plays an important but less essential role. The SxSx motif-deleted mutant was drastically attenuated and three single (S143A, S145A and C146A) mutants and a double (S143A/S145A) mutant exhibited various degrees of severity in their DNA-binding, DNA helicase and ATPase activities. Taken together, these results highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.

背景与目标： : UvrB亚基是UvrABC切口复合体的中心组成部分，在损伤识别，链切除和修复合成中起着关键作用。UvrB中存在的保守结构基序 (SxSx基序) 类似于超家族2解旋酶中的类似基序 (TxGx)，其功能尚未完全理解。为了阐明结核分枝杆菌UvrB (MtUvrB) 中SxSx (Ser143-Val144-Ser145-Cys146) 基序的重要性，构建并表征了MtUvrB亚基的不同变体。确实发现SxSx基序对于MtUvrB功能至关重要: 尽管该基序中的Ser143和Cys146残基对于MtUvrB功能至关重要，但Ser145起着重要但不那么重要的作用。SxSx基序缺失的突变体被急剧减毒，三个单个 (S143A，S145A和C146A) 突变体和一个双 (S143A/S145A) 突变体在其DNA结合，DNA解旋酶和ATPase活性方面表现出不同程度的严重程度。总之，这些结果突显了迄今为止SxSx基序在UvrB催化活性中的作用。

BACKGROUND & AIMS: :Transcriptional co-activator with PDZ-binding motif (TAZ) is a key downstream effector of the Hippo tumor-suppressor pathway. The functions of TAZ in the kidney, especially in tubular epithelial cells, are not well-known. To elucidate the adaptive expression, protective effects on kidney injury, and signaling pathways of TAZ in response to acute kidney injury (AKI), we used in vitro (hypoxia-treated human renal proximal tubular epithelial cells [RPTECs]) and in vivo (mouse ischemia-reperfusion injury [IRI]) models of ischemic AKI. After ischemic AKI, TAZ was up-regulated in RPTECs and the renal cortex or tubules. Up-regulation of TAZ in RPTECs subjected to hypoxia was controlled by IκB kinase (IKK)/nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) signaling. TAZ overexpression attenuated hypoxic and oxidative injury, inhibited apoptosis and activation of p38 and c-Jun N-terminal kinase (JNK) proteins, and promoted wound healing in an RPTEC monolayer. However, TAZ knockdown aggravated hypoxic injury, apoptosis, and activation of p38 and JNK signaling, delayed wound closure of an RPTEC monolayer, and promoted G0/G1 phase cell-cycle arrest. Chloroquine and verteporfin treatment produced similar results to TAZ overexpression and knockdown in RPTECs, respectively. Compared with vehicle-treated mice, chloroquine treatment increased TAZ in the renal cortex and tubules, improved renal function, and attenuated tubular injury and tubular apoptosis after renal IRI, whereas TAZ siRNA and verteporfin decreased TAZ in the renal cortex and tubules, deteriorated renal failure and tubular injury, and aggravated tubular apoptosis. Our findings indicate the renoprotective role of tubular TAZ in ischemic AKI. Drugs augmenting (e.g., chloroquine) or suppressing (e.g., verteporfin) TAZ in the kidney might be beneficial or deleterious to patients with AKI.

背景与目标： : 具有PDZ结合基序 (TAZ) 的转录共激活因子是Hippo肿瘤抑制途径的关键下游效应子。TAZ在肾脏中的功能，尤其是在肾小管上皮细胞中的功能尚不为人所知。阐明TAZ对急性肾损伤 (AKI) 的适应性表达，对肾损伤的保护作用以及信号通路。我们使用了缺血AKI的体外 (缺氧处理的人肾近端肾小管上皮细胞 [RPTECs]) 和体内 (小鼠缺血再灌注损伤 [IRI]) 模型。缺血AKI后，TAZ在rptec和肾皮质或肾小管中上调。缺氧的rptec中TAZ的上调受活化b细胞 (NF-κ B) 信号的i κ B激酶 (IKK)/核因子 κ 轻链增强子控制。TAZ过表达减轻了缺氧和氧化损伤，抑制了p38和c 6月N端激酶 (JNK) 蛋白的凋亡和活化，并促进了RPTEC单层的伤口愈合。然而，TAZ敲低加重了缺氧损伤，凋亡以及p38和JNK信号的激活，延迟了RPTEC单层的伤口闭合，并促进了G0/G1期细胞周期阻滞。氯喹和verteporfin处理分别产生了与RPTECs中TAZ过表达和敲低相似的结果。与赋形剂治疗的小鼠相比，氯喹治疗可增加肾皮质和肾小管中的TAZ，改善肾功能，减轻肾IRI后肾小管损伤和肾小管凋亡，而TAZ siRNA和verteporfin降低肾皮质和肾小管中的TAZ，恶化肾衰竭和肾小管损伤，并加重肾小管凋亡。我们的发现表明肾小管TAZ在缺血性AKI中的肾脏保护作用。在肾脏中增加 (例如氯喹) 或抑制 (例如verteporfin) TAZ的药物可能对AKI患者有益或有害。

BACKGROUND & AIMS: :Identification of functional motifs in a DNA sequence is fundamentally a statistical pattern recognition problem. Discriminant analysis is widely used for solving such problems. This paper will review two basic parametric methods: LDA (linear discriminant analysis) and QDA (quadratic discriminant analysis). Their usage in recognition of splice sites and exons in the human genome will be demonstrated.

背景与目标： : DNA序列中功能基序的识别从根本上说是一个统计模式识别问题。判别分析被广泛用于解决此类问题。本文将回顾两种基本的参数方法: LDA (线性判别分析) 和QDA (二次判别分析)。将证明它们在识别人类基因组中的剪接位点和外显子中的用途。

BACKGROUND & AIMS: :Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) are global epidemic public health problems with pathogenesis incompletely understood. Hepatocyte excessive apoptosis is a significant symbol for NAFLD/NASH patients, and therefore anti-apoptosis therapy could be used for NAFLD/NASH treatment. Up-regulation of BCL-2 has been found to be closely related with anti-apoptosis. BCL-2 gene promoter region has a C-rich sequence, which can form i-motif structure and play important role in regulating gene transcription. In this study, after extensive screening and evaluation, we found that acridone derivative A22 could up-regulate BCL-2 transcription and translation in vitro and in cells through selective binding to and stabilizing BCL-2 gene promoter i-motif. Our further experiments showed that A22 could reduce hepatocyte apoptosis in NAFLD/NASH model possibly through up-regulating BCL-2 expression. A22 could reduce inflammation, endoplasmic reticulum stress and cirrhosis in high-fat diet-fed mice liver model. Our findings provide a potentially new approach of anti-apoptosis for NAFLD/NASH treatment, and A22 could be further developed as a lead compound for NAFLD/NASH therapy. Our present study first demonstrated that gene promoter i-motif could be targeted for gene up-regulation for extended treatment of other important diseases besides cancer.

背景与目标： 非酒精性脂肪性肝病 (NAFLD)/非酒精性脂肪性肝炎 (NASH) 是全球流行的公共卫生问题，其发病机理尚未完全了解。肝细胞过度凋亡是NAFLD/NASH患者的重要标志，因此抗凋亡疗法可用于NAFLD/NASH治疗。已发现BCL-2的上调与抗凋亡密切相关。BCL-2基因启动子区具有富含C的序列，可形成i-基序结构，在基因转录调控中起重要作用。在这项研究中，经过广泛的筛选和评估，我们发现acridone衍生物A22可以通过选择性结合和稳定BCL-2基因启动子i-基序来上调体外和细胞中的BCL-2转录和翻译。我们进一步的实验表明，A22可能通过上调BCL-2表达来减少NAFLD/NASH模型中的肝细胞凋亡。A22可以减轻高脂饮食喂养小鼠肝脏模型的炎症，内质网应激和肝硬化。我们的发现为NAFLD/NASH治疗提供了一种潜在的抗凋亡新方法，A22可以进一步发展为NAFLD/NASH治疗的主要化合物。我们目前的研究首先证明了基因启动子i-motif可以作为基因上调的目标，以扩展治疗除癌症以外的其他重要疾病。

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。