Binding of the erlin1/2 complex to the third intralumenal loop of IP3R1 triggers its ubiquitin-proteasomal degradation

Long-term activation of inositol 1,4,5-trisphosphate receptors (IP₃Rs) leads to their degradation by the ubiquitin–proteasome pathway. The first and rate-limiting step in this process is thought to be the association of conformationally active IP₃Rs with the erlin1/2 complex, an endoplasmic reticulum–located oligomer of erlin1 and erlin2 that recruits the E3 ubiquitin ligase RNF170, but the molecular determinants of this interaction remain unknown. Here, through mutation of IP₃R1, we show that the erlin1/2 complex interacts with the IP₃R1 intralumenal loop 3 (IL3), the loop between transmembrane (TM) helices 5 and 6, and in particular, with a region close to TM5, since mutation of amino acids D-2471 and R-2472 can specifically block erlin1/2 complex association. Surprisingly, we found that additional mutations in IL3 immediately adjacent to TM5 (e.g., D2465N) almost completely abolish IP₃R1 Ca²⁺ channel activity, indicating that the integrity of this region is critical to IP₃R1 function. Finally, we demonstrate that inhibition of the ubiquitin-activating enzyme UBE1 by the small-molecule inhibitor TAK-243 completely blocked IP₃R1 ubiquitination and degradation without altering erlin1/2 complex association, confirming that association of the erlin1/2 complex is the primary event that initiates IP₃R1 processing and that IP₃R1 ubiquitination mediates IP₃R1 degradation. Overall, these data localize the erlin1/2 complex–binding site on IP₃R1 to IL3 and show that the region immediately adjacent to TM5 is key to the events that facilitate channel opening.     

Increased mTORC2 pathway activation in lymph nodes of iMCD‐TAFRO

Idiopathic multicentric Castleman disease (iMCD) is a rare and life‐threatening haematologic disorder involving polyclonal lymphoproliferation and organ dysfunction due to excessive cytokine production, including interleukin‐6 (IL‐6). Clinical trial and real‐world data demonstrate that IL‐6 inhibition is effective in 34–50% of patients. mTOR, which functions through mTORC1 and mTORC2, is a recently discovered therapeutic target. The mTOR inhibitor sirolimus, which preferentially inhibits mTORC1, has led to sustained remission in a small cohort of anti‐IL‐6‐refractory iMCD patients with thrombocytopenia, anasarca, fever, renal dysfunction and organomegaly (iMCD‐TAFRO). However, sirolimus has not shown uniform effect, potentially due to its limited mTORC2 inhibition. To investigate mTORC2 activation in iMCD, we quantified the mTORC2 effector protein pNDRG1 by immunohistochemistry of lymph node tissue from six iMCD‐TAFRO and eight iMCD patients who do not meet TAFRO criteria (iMCD‐not‐otherwise‐specified; iMCD‐NOS). mTORC2 activation was increased in all regions of iMCD‐TAFRO lymph nodes and the interfollicular space of iMCD‐NOS compared with control tissue. Immunohistochemistry also revealed increased pNDRG1 expression in iMCD‐TAFRO germinal centres compared with autoimmune lymphoproliferative syndrome (ALPS), an mTOR‐driven, sirolimus‐responsive lymphoproliferative disorder, and comparable staining between iMCD‐NOS and ALPS. These results suggest increased mTORC2 activity in iMCD and that dual mTORC1/mTORC2 inhibitors may be a rational therapeutic approach.     

LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine‐rich‐alpha‐2‐glycoprotein 1 (LRG‐1), that could promote fat graft survival through RAB31‐mediated inhibition of hypoxia‐induced apoptosis. We showed that LRG‐1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG‐1‐treated groups. Furthermore, we discovered that LRG‐1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG‐1‐mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG‐1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.     

Design,synthesis, and biological evaluation of novel carbazole derivatives as potent DNMT1 inhibitors with reasonable PK properties

The DNA methyltransferases (DNMTs) were found in mammals to maintain DNA methylation. Among them, DNMT1 was the first identified, and it is an attractive target for tumour chemotherapy. DC_05 and DC_517 have been reported in our previous work, which is non-nucleoside DNMT1 inhibitor with low micromolar IC₅₀ values and significant selectivity towards other S-adenosyl-_L-methionine (SAM)-dependent protein methyltransferases. In this study, through a process of similarity-based analog searching, a series of DNMT1 inhibitors were designed, synthesized, and evaluated as anticancer agents. SAR studies were conducted based on enzymatic assays. And most of the compounds showed strong inhibitory activity on human DNMT1, especially WK-23 displayed a good inhibitory effect on human DNMT1 with an IC₅₀ value of 5.0 µM. Importantly, the pharmacokinetic (PK) profile of WK-23 was obtained with quite satisfying oral bioavailability and elimination half-life. Taken together, WK-23 is worth developing as DNMT1-selective therapy for the treatment of malignant tumour.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

大肠杆菌共表达LeGGPS2、LePSY1和crtI基因合成番茄红素

利用大肠杆菌研究番茄红素的合成,不仅可以获得副产物少的高产菌株,而且可以探讨基因或基因簇的功能。文中将番茄LeGGPS2和LePSY1的cDNA序列,及欧文氏菌crtI的编码序列分别添加上核糖体结合位点后,以单独或组合的方式受控于T7启动子和终止子,在大肠杆菌菌株BL21(DE3)中进行表达和诱导番茄红素合成。结果显示,仅T7::crtI-LeGGPS2-LePSY1三价基因共表达时才能合成番茄红素,且将种子液以1∶50接种于含3%蔗糖的LB培养基(pH 6.8)中,于37℃摇8 h左右的对数生长后期加IPTG至80μmol/L,30℃诱导表达5 h的发酵条件下,获得2.124 mg/g DCW的番茄红素。该结果既验证了原核化的番茄LeGGPS2和LePSY1基因及与crtI基因协同作用的功能,又为在番茄质体中建立独立的番茄红素合成途径奠定了基础。     

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.     

Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.