Regulation of clpQ+Y+ (hslV+U+) Gene Expression in Escherichia coli

The Escherichia coli ClpYQ (HslUV) complex is an ATP-dependent protease, and the clpQ+Y+ (hslV+U+) operon encodes two heat shock proteins, ClpQ and ClpY, respectively. The transcriptional (op) or translational (pr) clpQ+::lacZ fusion gene was constructed, with the clpQ+Y+ promoter fused to a lacZ reporter gene. The clpQ+::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λclpQ+::lacZ+ (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH or/and rpoS mutants. Upon shifting the temperature up from 30 °C to 42 °C, the wild-type λclpQ+::lacZ+ (op) demonstrates an increased β-galactosidase (βGal) activity. However, the βGal activity of clpQ+::lacZ+ (op) was decreased in the rpoH and rpoH rpoS mutants but not in the rpoS mutant. The levels of clpQ+::lacZ+ mRNA transcripts correlated well to their βGal activity. Similarly, the expression of the clpQ+::lacZ+ gene fusion was nearly identical to the clpQ+Y+ transcript under the in vivo condition. The clpQm1::lacZ+, containing a point mutation in the -10 promoter region for RpoH binding, showed decreased βGal activity, independent of activation by RpoH. We conclude that RpoH itself regulates clpQ+Y+ gene expression. In addition, the clpQ+Y+ message carries a conserved 71 bp at the 5’ untranslated region (5’UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQm2Δ40::lacZ+, with a 40 bp deletion in the 5’UTR, showed a decreased βGal activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ+Y+ message

Ample Pairs

We show that the ample degree of a stable theory with trivial forking is preserved when we consider the corresponding theory of belles paires, if it exists. This result also applies to the theory of $H$-structures of a trivial theory of rank $1$.Comment: Research partially supported by the program MTM2014-59178-P. The second author conducted research with support of the programme ANR-13-BS01-0006 Valcomo. The third author would like to thank the European Research Council grant 33882

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities

Investigation of clpQ+clpY+ and gspS+ in Escherichia coli: gene regulation and substrate recognition

熱休克蛋白普遍存在於生物體內，作用為幫助生物抵抗環境衝擊。本文針對大腸桿菌熱休克蛋白ClpYQ (HslUV)的基因調控及基質辨識深入研究。遇到環境衝擊時，大腸桿菌會發生熱休克反應，此時折疊錯誤的蛋白促使細胞內熱休克基因表現，這些表現常由調控蛋白sigma factor，如RpoH (或稱σ32) 所調控。本文使用大腸桿菌熱休克基因clpQ+clpY+的啟動子與報導基因lacZ建構轉錄融合基因clpQ+::lacZ (op)及轉譯融合基因clpQ+::lacZ (pr)，利用噬菌體λRS45將融合基因clpQ+::lacZ帶入野生株及rpoS -，rpoH -，rpoH -rpoS -突變株後，偵測β-galactosi dase活性以瞭解clpQ+::lacZ的表現。當溫度由30℃升至42℃時，野生株及rpoS -突變株的β-galactosidase活性上升，但rpoH -及rpoH -rpoS -突變株則未見此現象。由β-galactosidase 活性分析與北方點墨法分析結果，可知clpQ+::lacZ轉錄的mRNA訊號強度與β-galactosidase活性成正比，且clpQ+::lacZ與clpQ+clpY+的表現相似。針對clpQ+clpY+啟動子上σ32 (rpoH) 可辨識的保守序列做C→T點突變，此突變使融合基因clpQm(c→t)::lacZ無法被σ32活化，β-galactosidase活性下降。經由遺傳分析結果證實，大腸桿菌clpQ+clpY+的啟動子可被σ32辨識。此外，clpQ+clpY+operon的五端未轉譯區域 (5’-UTR)，亦即轉錄起始處 (transcription start site) 與起始密碼 (start codon) 之間，長度為71 bp，此五端未轉譯區域帶有一段inverted repeat sequence (IR序列) 5’ CC CCGTAC TTTTGTACGGGG 3’，此IR序列普遍存在於腸道菌的clpQ+clpY+五端未轉譯區域中。藉由刪除此段IR序列，並與lacZ融合，分析融合基因clpQm2△40bp::lacZ的β-galactosidase 活性，以及此段序列缺失對ClpQ與ClpY間交互作用的影響，顯示IR所形成的stem-loop二級結構在clpQ+clpY+表現時，具有穩定mRNA的效果，本研究為ATP依賴型蛋白酶 (ATP-dependent protease)中，首次發現5’ stem-loop結構具有穩定下游mRNA的功能。在基質辨識的研究部分，ClpYQ以六元環方式組合，其中ClpY負責基質辨識，打開基質結構，並傳送至ClpQ進行分解。ClpY可分為三個作用區(domain)，N-terminal domain，I-intermediated domain及C-terminal domain，N domain具有ATPase的功能，C domain則與self-oligomerization及ClpQ的蛋白酶活性相關。本文使用酵母菌雙雜交系統，得知ClpY的I domain負責基質辨識，C domain則可與ClpQ作用，而I domain中的loop L2(175-209 aa)除了與基質結合外，並與後續的基質傳遞及分解相關。另外，本文亦研究大腸桿菌的酵素glutathionylspermidine synthetase之基因gspS，由於病原蟲Trypanosomatida會造成人類昏睡，發炎及死亡，而酵素TryS為此種病原蟲所特有，在人類細胞中不存在，所以TryS的研究對治療Trypanosomatida所造成的疾病極為重要，本文針對大腸桿菌中與TryS構造及功能皆相似的酵素GspS，其基因表現之調控做一初步的探討。大腸桿菌的gspS 長1860 bp，產物為glutathionylspermidine synthetase (GspS)，共有619個胺基酸，是一種具有雙重功能的酵素 (bifunctional enzyme)，可執行GSH和spermidine之間醯胺鍵的合成與分解。本研究確認gspS為一單獨的轉錄單位而非以操縱子形式存在，且GspS起始密碼上游序列具有啟動子功能，而在in vivo情況下，H2O2與BaeR都可誘導gspS之表現。(1) Heat shock responses are typically observed in E. coli. Upon heat shock, protein misfolding leads to a cascade of intracellular protein synthesis, usually dependent on a sigma factor, i.e., σ32, for their gene expression. In this study, the transcriptional (op) or translational (pr) clpQ+::lacZ fusion gene was constructed, with the clpQ+clpY+ promoter fused to a lacZ reporter gene. The clpQ+::lacZ (op or pr) fusion gene was each crossed into lambda phage. The λclpQ+::lac (op), a transcriptional fusion gene, was used to form lysogens in the wild-type, rpoH - or/and rpoS - mutants. Upon shifting the temperature up from 30 ℃ to 42 ℃, the wild-type λclpQ+::lacZ(op) demonstrates an increased β-galactosidase activity. However, the β-galactosidase activity of clpQ+::lacZ(op) was decreased in the rpoH - and rpoH -rpoS - mutants but not in the rpoS - mutant. The levels of clpQ+::lacZ mRNA transcripts correlated well to their β-galactosidase activity. Similarly, the expression of the clpQ+::lacZ gene fusion was nearly identical to the clpQ+clpY+ transcript under the in vivo condition. The clpQm(c→t)::lacZ, containing a C to T point mutation in the -10 promoter region for RpoH binding, showed decreased β-galactosidase activity, independent of activation by RpoH. Thus, through a genetic analysis, the clpQ+clpY+ promoter is in vivo recognized by σ32. The transcriptional start point of the clpQ+clpY+ gene lies 71 bases upstream from the clpQ+ start codon. An untranslated region (UTR) upstream of this mRNA contains a 20 bp inverted repeat (IR) sequence 5’CCCCGTACTTTTGTAC GGGG3’, which is unique for the clpQ+clpY+ operon. In addition, from the wild bacterial genome, the 5’UTR of clpQ+clpY+ also exists in other bacterial species. The clpQ+clpY+ message carries a conserved 71 bp at the 5’ untranslated region (5’UTR) that is predicted to form the stem-loop structure by analysis of its RNA secondary structure. The clpQm2△40bp::lacZ, with a 40 bp deletion in the 5’UTR, showed a decreased β-galactosidase activity. In addition, from our results, it is suggested that this stem-loop structure is necessary for the stability of the clpQ+clpY+ message. It is noteworthy that this is the first example in the ATP dependent protease to demonstrate that the 5’ stem-loop structure itself participates in the stability of its downstream mRNA. (2) Regarding ClpY substrate recognition study, in the presence of ATP, the ClpYQ complex forms an active protease with an Y6Q6Q6Y6 configuration. ClpY binds, unfolds, and transfers the substrates outside the cylinder into a catalytic core where ClpQ degrades the substrates. The ClpY molecule is divided into three domains: the N-terminal domain, I-intermediate domain and C-terminal domain. The N domain has an ATPase activity, and the C domain is responsible for self-oligomerization of ClpY. Using the yeast two-hybrid system, we show that domain I of ClpY is responsible for recognition of its natural substrates while domain C is necessary for association with ClpQ. The loop 175-209 aa plays a role in substrate tethering. (3) In addition to clpQ+clpY+, gspS+ in Escherichia coli is included in this study. Parasitic Trypanosoma species cause serious tropical diseases such as kala-azar, African sleeping sickness, and Chagas diseases. Trypanothione synthetase (TryS) is a protein unique to Trypanosoma. However, Escherichia coli produce only the metabolic intermediate GspdSH by enzyme GspS, but not trypanothione. Evolutionary, TryS and GspS share the similarly functional domains. The gspS of E. coli, encoding a bifunctional enzyme GspS of 619 amino acids, is a gene with 1860 bp. GspS is responsible for the activities of amidase and synthetase between GSH and spermidine.In this study, we showed that gspS in E. coli is an unique transcriptional unit, and the singular promoter was present in the upstream region of the GspS structural gene. In addition, the gspS promoter is in vivo induced by H2O2 and BaeR.目錄謝 i要 iibstract iv錄 vii目錄 x目錄 xi發表文章 xii一章 前言 1.1 clpQ+clpY+研究源起 1.2 熱休克蛋白 (heat shock proteins) 與ATP依賴型蛋白酶 (ATP-dependent protease) 簡介 1.3 ClpYQ蛋白酶之簡介 2.3.1 clpQ+clpY+操縱子 (operon) 的發現及命名 2.3.2 ClpYQ為熱休克蛋白的證據 3.4 ClpYQ為ATP依賴型蛋白酶之研究 3.4.1 ClpY為ATPase之研究 3.4.2 ClpY具chaperone功能之研究 4.4.3 ClpYQ蛋白酶為Threonine蛋白酶 5.4.4 ClpYQ蛋白酶為ATP依賴型蛋白酶 6.5 ClpYQ蛋白複合體的結構 7.5.1 ClpQ及ClpY可形成複合體 7.5.2 由蛋白分子量推測ClpYQ蛋白複合體結構 7.5.3 由電顯影像分析ClpYQ蛋白複合體結構 8.6 ClpYQ蛋白酶基質辨識區域之相關研究 9.7 ClpYQ蛋白酶基質之研究 10.7.1 ClpYQ蛋白酶可分解異蛋白 (abnormal proteins) 10.7.2 ClpYQ蛋白酶可分解SulA 10.7.3 ClpYQ蛋白酶可分解RcsA 12.8 clpQ+clpY+實驗目的 12.9 gspS+研究源起 13.10 GspdSH簡介 14.10.1 GspdSH組成物質: GSH及spermidine 14.10.2 大腸桿菌中GspdSH及其合成酵素GspS相關之研究 15.11 雙成份控制系統BaeSR之研究 17.12 gspS+實驗目的 19二章 調控蛋白sigma factors對clpQ+clpY+表現的影響 21.1 摘要 21.2 材料與方法 21.2.1 建立clpQ+clpY+上游啟動子區域 ( promoter region ) 表現系統 21.2.2 β-galactosidase活性分析 24.2.3 北方點墨法 (Northern blotting) 25.2.4 引子延伸實驗 (primer extension) 26.3 結果 28.3.1 clpQ+::lacZ轉錄及轉譯融合基因的表現 28.3.2 RpoH對clpQ+::lacZ基因表現的影響 28.3.3 clpQm1::lacZ啟動子上的點突變對clpQm1::lacZ基因表現的影響 31.3.4 clpQm(c→t)::lacZ及clpQm1::lacZ啟動子上的點突變不影響轉錄起始點 32.3.5 clpQ+clpY+ 操縱子 (operon) 的mRNA表現與clpQ+::lacZ的β-galactosidase活性 32.3.6 不同蛋白酶缺失對clpQ+::lacZ的影響 33.4 討論 34三章 clpQ+clpY+ 啟動子五端未轉譯區域 (5’-UTR) 對ClpQ表現的影響 36.1 摘要 36.2 材料與方法 36.2.1 建立AC3112(cpsB::lacZ)/pBAD33-clpQ/pBAD24-clpY表現系統 36.2.2 AC3112/pBAD33-clpQ/pBAD24-clpY中，ClpYQ對兩種基質RcsA及SulA的分解能力測試 38.2.3 β-galactosidase活性分析 39.2.4 北方點墨法 (Northern blotting) 39.2.5 mRNA穩定性測試 41.2.6 西方點墨法 (Western blotting) 41.2.7 預測RNA二級結構所使用的網站 43.3 結果 43.3.1 clpQ+clpY+ 啟動子的五端未轉譯區域 (5’-UTR) 對clpQ+::lacZ表現的影響 43.3.2 五端未轉譯區域 (5’-UTR) 中的IR序列對clpQ+ clpY+表現的影響 44.3.3 IR序列所形成的stem-loop結構對clpQ+ clpY+表現的影響 46.4 討論 47四章 ClpY基質辨識位置之探討 49.1 摘要 49.2 材料與方法 49.2.1 酵母菌雙雜交系統的菌株、載體及培養基 49.2.2 leu2 expression : 生長測試 50.2.3 lacZ expression：X-gal測試 50.2.4 lacZ expression：b-galactosidase活性分析 51.2.5 ClpY及其突變分解基質之偵測 52.3 結果 52.3.1 ClpY基質辨識：ClpY△I+7Gly及ClpY△L1, △L2與SulA之間沒有交互作用 52.3.2 ClpY基質辨識： loopL2上的點突變ClpYL199Q造成ClpYL199Q/ClpQ無法分解基質 53.3.3 ClpY基質辨識：ClpY的基質辨識與loopL2上的疏水性胺基酸密切相關 53.3.4 ClpY與ClpQ之交互作用: ClpY△L1，ClpY△L2，ClpY△L1, △L2及ClpY△I+7Gly能與ClpQE61C結合，ClpY△c，ClpYX則否 54.4 討論 55五章 大腸桿菌gspS基因表現之調控 56.1 摘要 56.2 材料與方法 56.2.1 建立gspS上游啟動子區域表現系統 56.2.2 β-galactosidase活性分析 58.2.3 預測啟動子、密碼子及操縱子所使用的網站 58.3 結果 59.3.1 gspS 上游序列之探討 59.3.2 影響gspS表現的因素 64.4 討論 67考文獻 69目錄一、使用於clpQ+clpY+之研究的菌株 76二、使用於clpQ+clpY+之研究的載體與噬菌體 77三、使用於clpQ+clpY+之研究的引子 78四、使用於gspS之研究的菌株 79五、使用於gspS之研究的載體與噬菌體 80六、使用於gspS之研究的引子 81七、操縱子預測結果 82八、SG20250/pRS415-gspS-lacZ(op)與SG20250/λRS45-gspS-lacZ的β-galactosidase活性分析 83九、BaeR對gspS-lacZ表現之影響 84十、BaeR與H2O2對gspS-lacZ表現的影響 85目錄一、clpQ+ clpY+操縱子的啟動子區域之序列圖示 86二、不同長度的轉錄及轉譯融合基因clpQ+clpY+::lacZ之活性 87三、wt, rpoS -, rpoH -, rpoS –rpoH –四種菌株內，42 ℃對clpQ+::lacZ的熱誘導 88四、42 ℃對clpQ+::lacZ熱誘導受啟動子點突變(C→T)的影響 89五、啟動子點突變 (A→C) 對clpQm1::lacZ表現的影響 90六、clpQ+啟動子點突變對轉錄起始點(+1)沒有影響 91七、clpQ+::lacZ 與clpQ+clpY+在30℃時的mRNA表現 92八、蛋白酶缺失對clpQ+::lacZ表現的影響 93九、去除5’-UTR對clpQm2△40bp::lacZ表現的影響 94十、五端未轉譯區域 (5’-UTR) 中的IR序列對clpQ+ clpY+表現的影響 95十一、IR序列對ClpQ/ClpY 交互作用之影響 96十二、IR 序列所形成的stem-loop結構對clpQ+ clpY+表現的影響 97十三、不同菌種間clpQ+ clpY+五端未轉譯區域(5’-UTR)保守性序列之比對 98十四、ClpY三個主要作用區示意圖 99十五、ClpY的loop L1與loop L2與SulA間交互作用之結果。 100十六、四組ClpY點突變M187I，A188S，L199Q，N205K與基質結合及分解基質能力 101十七、三組ClpY突變I186N，E193L,E194L，Q198L,Q200L與基質結合及分解基質能力 102十八、帶有ClpQE61C/ClpY融合蛋白的EGY48/p8op-lacZ，其Leu2的表現 103十九、gspS 上游區域之啟動子預測 104二十、gspS上游區域的編碼區預測 105二十一、啟動子與編碼區預測結果比較 106二十二、編碼區與操縱子預測結果比較 107二十三、gspS-lacZ融合基因之建構 108二十四、H2O2對gspS-lacZ表現的影響 109二十五、BaeR與H2O2對HY1002(gspS-lacZ)的生長狀況之影響 110二十六、H2O2對HY1002(gspS-lacZ)的生長狀況之影響 111圖一、大腸桿菌 ClpYQ 結構圖 11

Craniofacial Growth and Asymmetry in Newborns: A Longitudinal 3D Assessment

Objective: To evaluate the development of the craniofacial region in healthy infants and analyze the asymmetry pattern in the first year of life. Methods: The participants were grouped by sex and age (1, 2, 4, 6, 9, and 12 months) to receive three-dimensional (3D) photographs. Stereoscopic craniofacial photos were captured and transformed into a series of craniofacial meshes in each group. The growth patterns of the anthropometric indices and the degree of craniofacial asymmetry were measured, and average craniofacial meshes and color-asymmetry maps with craniofacial asymmetry scores were calculated. Results: A total of 373 photographs from 66 infants were obtained. In both genders, the highest and lowest growth rates for all anthropometric indices were noted between 1 and 2 months and between 9 and 12 months, respectively. Overall, male infants had higher anthropometric indices, head volume, and head circumference than female infants. The craniofacial asymmetry score was presented with a descending pattern from 1 to 12 months of age in both sex groups. Both sex groups showed decreased left-sided laterality in the temporal-parietal-occipital region between 1 and 4 months of age and increased right frontal-temporal prominence between 6 and 12 months of age. Conclusions: A longitudinal evaluation of the craniofacial growth of healthy infants during their first year of life was presented

Dengue virus enhances thrombomodulin and ICAM-1 expression through the macrophage migration inhibitory factor induction of the MAPK and PI3K signaling pathways.

Dengue virus (DV) infections cause mild dengue fever (DF) or severe life-threatening dengue hemorrhagic fever (DHF). The mechanisms that cause hemorrhage in DV infections remain poorly understood. Thrombomodulin (TM) is a glycoprotein expressed on the surface of vascular endothelial cells that plays an important role in the thrombin-mediated activation of protein C. Prior studies have shown that the serum levels of soluble TM (sTM) and macrophage migration inhibitory factor (MIF) are significantly increased in DHF patients compared to levels in DF patients or normal controls. In this study, we investigated how MIF and sTM concentrations are enhanced in the plasma of DHF patients and the potential effect of MIF on coagulation through its influence on two factors: thrombomodulin (TM) and intercellular adhesion molecule-1 (ICAM-1) in endothelial cells and monocytes. Recombinant human macrophage migration inhibitory factor (rMIF) was used to treat monocytic THP-1 cells and endothelial HMEC-1 cells or primary HUVEC cells. The subsequent expression of TM and ICAM-1 was assessed by immunofluorescent staining and flow cytometry analysis. Additionally, the co-incubation of THP-1 cells with various cell signaling pathway inhibitors was used to determine the pathways through which MIF mediated its effect. The data provided evidence that severe DV infections induce MIF expression, which in turn stimulates monocytes or endothelial cells to express TM and ICAM-1 via the Erk, JNK MAPK and the PI3K signaling pathways, supporting the idea that MIF may play an important role as a regulator of coagulation

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer’s vesicle morphogenesis and spaw expression

Abstract Background Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer’s vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Methods Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer’s vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Results Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18–22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer’s vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. Conclusions Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer’s vesicle morphogenesis and spaw expression in the left LPM

Characterization of the Escherichia coli ClpY (HslU) Substrate Recognition Site in the ClpYQ (HslUV) Protease Using the Yeast Two-Hybrid System ▿

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ