Heat-shock cognate 70 is required for the activation of heat-shock factor 1

REVIEWChaperone-mediated autophagy:machinery,regulation and biological consequencesWenming Li •Qian Yang •Zixu MaoReceived:12July 2010/Revised:7October 2010/Accepted:8October 2010/Published online:26October 2010ÓSpringer Basel AG 2010Abstract Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways,i.e.,macroautophagy,micro-autophagy and chaperone-mediated autophagy (CMA).In this review,we focus on CMA,a type of autophagy distinct from the other two autophagic pathways owing to its selectivity,saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly deliv-ered into the lysosomal lumen via specific receptors.CMA participates in quality control to maintain normal cell functions by clearing ‘‘old’’proteins and provides energy to cells under nutritional stress.Deregulation of CMA has recently been shown to underlie some diseases,especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor.Therefore,targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.Keywords CMA ÁLysosome ÁHsc70ÁLamp2a ÁAutophagy ÁUnibiquitinIntroductionMaintaining a balance between protein synthesis and deg-radation is absolutely essential for proper cellular function,homeostasis and survival in a changing extracellularenvironment.The two major protein degradation mecha-nisms in eukaryotes are the ubiquitin-proteasome system (UPS)and the autophagy-lysosome system [1,2].Collec-tively,they (1)exert quality control by clearing damaged or incorrectly synthesized proteins,(2)recycle proteins that are no longer needed into constitutive amino acid compo-nents,(3)mount the cellular defense by breaking down components of various invading pathogens,and (4)regu-late cellular response to stress or the alteration in extracellular micro-environments,assisting cells in adapt-ing to new changes [3,4].The UPS is a multi-subunit protease complex in the cytosol that permits entry and subsequent degradation of proteins tagged with one or more covalently bound ubiquitin molecules [5].Ubiquitin is a small protein highly conserved from yeast to man.It was first described in the context of protein degradation,but later shown to participate in regulation of other cellular processes,such as endocytosis,signal transduction and DNA repair.Conjugation of ubiquitin is a complex reaction that requires E1,E2and E3enzymes,and leads to the for-mation of an isopeptide bond between the C-terminal glycine of ubiquitin and the 3-amino group of a lysine residue of the substrate protein [6,7].Subunits of the regulatory complex of the proteasome recognize the ubiquitin tag,remove it and mediate the unfolding of the substrates required to gain access to the catalytic region of the proteasome barrel.With certain exceptions,most proteasome substrates have short half-lives [6,8].Inter-estingly,recent studies suggest that ubiquitin and lysosomal pathways are linked.For example,new evi-dence suggests that attachment of ubiquitin to various cellular cargos not only constitutes a degradation signal for proteasome,but also serves to signal for cargo removal by lysosomal system via autophagy [2,9].W.Li ÁQ.Yang ÁZ.Mao (&)Departments of Pharmacology and Neurology,Emory University School of Medicine,Atlanta,GA 30322,USAe-mail:zmao@Cell.Mol.Life Sci.(2011)68:749–763DOI 10.1007/s00018-010-0565-6Cellular and Molecular Life SciencesAutophagy is a conserved cellular ‘‘self-eating’’process that involves sequestration and delivery of cytosolic components to the lysosome for degradation and recycling [10].This evolutionarily conserved process can be cate-gorized into three classes depending on their respective sequestration and delivery mechanism (Fig.1)[11].In macroautophagy,a double-membrane vesicle termed the autophagosome is formed to engulf long-lived proteins and organelles.The autophagosome is subsequently fused with a lysosome,releasing its cargo for degradation by lyso-somal hydrolases.The resultant nucleotides,amino acids and fatty acids are eventually recycled back into the cytosol for reuse.In microautophagy,the sequestration of cytosolic content is facilitated by direct invagination or exvagination of lysosomal membrane,and subsequent budding of the invaginated vesicles into the lysosomal lumen releases the sequestered cytosolic material.In con-trast to the vesicle-mediated substrate delivery of macro and microautophagy,chaperone-mediated autophagy (CMA)targets and delivers substrate proteins directly across the lysosomal membrane via the specific receptor [9,12].Only proteins containing a consensus peptide sequence are recognized by a chaperone complex.This CMA sub-strate-chaperone complex locates to the lysosome through interaction with the lysosome receptor and translocates the substrate across the lysosomal membrane with the assis-tance of lysosomal chaperones on the lumenal side [12,13].In this review,we first describe the machinery of CMA,summarize the regulation mechanisms involved in activation of CMA,and finally discuss the biological consequences of CMA under various physiological and pathological conditions,and relevant therapeutic strategies via regulating CMA.History of CMAIn the early 1980s,Professor Dice’s group first reported that radiolabeled RNase A introduced into the cytoplasm of human fibroblasts by using erythrocyte-mediated microin-jection or osmotic lysis of pinosomes was degraded with a half-life of approximately 90h in the presence of serum,whereas in response to serum deprivation its rate of deg-radation was enhanced 1.6-fold [14].This enhanced breakdown following serum withdrawal was highly selec-tive and based on a feature present within the N-terminal 20amino acids of RNase A [15].During subsequent years,the essential motifs related to KFERQ were identified in proteins serving as substrates of this selective degradation pathway,which is referred to as the selective pathway for degradation of cytosolic proteins by lysosomes [16,17].In 1989,a 73-kDa heat shock cognate protein (Hsc73,now commonly referred toas Hsc70)was found to bind to KFERQ-like regions in intracellular proteins that are tar-geted for lysosomal degradation in response to serum deprivation [18].In 1994,isolated rat liver lysosomes were used to probe the selective binding and uptake of RNase A and glyceraldehyde-3-phosphate dehydrogenase.Their uptake and degradation by lysosomes were progressively activated in rat liver by starvation [19].In 1996,Lamp2a,also named Lgp96(lysosomal glycoprotein of 96kDa),was identified as a receptor for the selective import and degradation of proteins within lysosomes [20].In 1997,an intralysosomal Hsc73was determined to be required for the selective pathway of lysosome-mediated protein deg-radation [21].Since 2000,this pathway has been formally named chaperone-mediated autophagy (CMA)[22,23].Throughout the subsequent decade,new substrates,moreFig.1Autophagy refers to the conserved degradation of intracellular components by lysosomes.In mammals,three types of autophagy have been described:macroautophagy,microautophagy and CMA.Adapted from [11]with permission750W.Li et al.detailed machinery,new components,physiological roles and associated diseases for CMA have been extensively investigated and elucidated [3].Machinery of CMACMA uses the unique and distinctive machinery from the other two autophagy pathways to carry out this process [3,24].The basic machinery consists of at least three types of proteins,including (1)chaperone proteins,which are responsible for recognizing substrates based on their spe-cific motifs and delivering them to lysosomes,(2)receptor proteins,which bind and transport/pull substrates into lysosome lumens,and (3)substrate proteins,which are a subset of soluble cytosolic proteins containing specific motifs related to KFERQ.Following activation of CMA,these three subsets of proteins collaborate and complete the process (Fig.2)[3,25].Workshop—lysosomeLysosomes are the primary catabolic compartment of eukaryotic cells.The name lysosome derives from the Greek words lysis,which means dissolution or destruction,and soma,which means body [24].Lysosomes were dis-covered by the Belgian cytologist Christian de Duve in 1949[26].They are created by the addition of hydrolyticenzymes to early endosomes from the Golgi apparatus [24].The membrane around a lysosome allows the digestive enzymes to work at pH 5.1–5.5,which is optimal to these acidic hydrolases.Lysosomes fuse with vacuoles and dis-pense their enzymes within digesting the contents [27].A healthy cell is dependent on the proper targeting of newly synthesized lysosomal proteins.Two classes of proteins are essential for the function of lysosomes:soluble lysosomal hydrolases (i.e.,acid hydrolases)and integral lysosomal membrane proteins (LMPs)[24].Lysosomal acidic pH and the large variety of hydrolases present in the lysosomal lumen (including proteases,lipases,glycosi-dases and nucleases)confer upon this organelle its high capacity of degradation and mediate complete breakdown of all types of molecules.In addition to bulk degradation,lysosomal hydrolases are involved in antigen processing,degradation of the extracellular matrix and initiation of apoptosis [24,28].LMPs reside mainly in the lysosomal limiting membrane and have diverse functions,including acidification of the lysosomal lumen,protein import from the cytosol,membrane fusion and transport of degradation products to the cytoplasm [29].The main LMPs are lyso-some-associated membrane protein 1and 2(Lamp1and 2),lysosomal integral membrane protein 2and tetraspanin CD63[24].Substrates can reach lysosomes via heteroph-agy,in which the cargo to be degraded originates at the plasma membrane or extracellularly,or via autophagy,for cargo located in the cytosol [3,24,30].Fig.2The diagram ofproposed mechanisms of CMA:a Hsc70with co-chaperones recognizes a KFERQ-related peptide in cytosolic substrate proteins;b the complex binds to the Lamp2a receptor on the lysosomal membrane;c the substrate protein is unfolded before traversing the lysosomal membrane;d lys-Hsc70pulls the substrate into the lysosome matrix;e the substrate protein is degraded by lysosomal proteases;f the Hsc70-cochaperone complex is released from the lysosomal membrane;g Hsc70is available to bind to another CMA substrate.Adapted from [25]with permissionBasics of chaperone-mediated autophagy 751Chaperones—Hsc70and partnersHsc70,a73-kDa protein,is the constitutive member of the heat shock protein70family of chaperone[19,25].Hsc70s have been found to be involved in many cellular processes including dissociation of clathrin and assembly proteins [25,31].Binding of Hsc70to substrate proteins is regu-lated by ATP binding and hydrolysis,and the ADP-bound form of Hsc70has the highest affinity for protein substrates for CMA[19,32].Hsc70is located in the cytosol or in the lumen of lysosomes.Cytosolic Hsc70(cyt-Hsc70)can recognize a peptide sequence including the KFERQ motif in CMA substrate proteins and aid in their transport to the lysosomal receptor[3,19].Cyt-Hsc70docked to the lysosomal membrane helps to unfold substrate proteins,a necessary step for their entry into lysosomes[33].Other co-chaperones interact with Hsc70and regulate its activities.Hsp40may activate the ATPase activity of Hsc70to facilitate substrate binding;and the Hsp70-interacting protein(Hip)stimulates the assembly of Hsc70 with Hsp40and the protein substrate[25].Cell division cycle48(Cdc48)can also enhance the activity of Hsc70-Hsp40complexes[32,34].Hsc70-Hsp90organizing pro-tein acts as an adapter between Hsc70and Hsp90,which recognize unfolded regions within proteins and prevent substrate protein aggregation[25,32].There are co-chap-erones of Hsp90that may be in the molecular chaperone complex.Activator of Hsp90ATPase is a family of heat shock proteins that activates the ATPase activity of Hsp90 and thereby stimulates both protein binding and release [32,35].The Bcl2-associated athanogene1protein(Bag-1) was initially described as a co-chaperone of Hsc70that uncouples the ATPase cycle from substrate binding[36]. But several subsequent studies showed that it functions as a nucleotide exchange factor that stimulates substrate release [37].The carboxyl terminus of Hsc70-interacting protein (Chip),which can regulate protein refolding[38],acts as a chaperone-associated ubiquitin ligase to stim-ulate the degradation of Hsc70client proteins[39].The chaperone complex present on the cytosolic face of lyso-somal membranes is linked to Lamp2a by the substrate protein via a site that is different from sites of interaction for the molecular chaperone complex.Each of the com-ponents in the molecular chaperone complex is required for transport of substrates into the lysosome lumen[25,32].Both Hsc70and Hsp90have been found to be also present in the lysosomal lumen.Lysosomal-Hsc70(lys-Hsc70)is required for the CMA pathway[21].The more active population of lysosomes contains abundant lys-Hsc70,whereas the less active ones contain little Hsc70. The latter group of lysosomes can be made more active for CMA if they are allowed to take up Hsc70.A role for lys-Hsc70in the uptake of substrate proteins has been demonstrated in cultured confluent humanfibroblasts.It has been speculated that lys-Hsc70may be required to pull proteins into the lysosomal lumen because of analogous roles of Hsp70s in the translocation of proteins into the endoplasmic reticulum,mitochondria and chloroplasts [21,25].Nearly half of the lysosomal Hsp90s associate with the lumenal side of the lysosomal membrane where this chaperone may contribute to the stabilization of essential components of the translocation complex when it is organized into a multimeric structure[3,40].Receptor—Lamp2aIn1996,a lysosomal membrane glycoprotein Lgp96 (called Lamp2a in human)was identified as a receptor for binding and uptake of lysosome substrates[20].Lamp2a is one of the three splice variants of the Lamp2gene that gives rise to three single-span membrane proteins, Lamp2a,b and c.These variants all have a common highly N-glycosylated lumenal region,but possess different transmembrane and C-terminal cytosolic tail regions. Lamp2a has a short cytosolic tail(GLKRHHTGYEQF)to which CMA substrate proteins bind[25].The positively charged residues in the Lamp2a cytosolic tail are important for binding of substrate proteins.However,the specific amino acids on the CMA substrate proteins required for receptor binding remain elusive[41].Binding of substrate proteins to Lamp2a is a rate-limiting step for the CMA process,and overexpression of Lamp2a in Chinese hamster ovary cells can increase CMA mp2a is not limited to acting as a classic receptor.Increasing evidence shows that Lamp2a is involved in many other aspects of the CMA process such as substrate translocation[3,25,40]. Lamp2deficiency in mice causes extensive accumulation of autophagic vacuoles in many tissues[42].Contrary to the proposed receptor function of Lamp2a in CMA,con-fluent mouse embryonicfibroblasts deficient in lamp2 appear to have normal levels of CMA-associated lysosomal proteolysis after prolonged serum withdrawal,suggesting that Lamp2a may not be the only receptor for CMA [29,43].Substrates—KFERQ motifSubstrates for CMA are defined by an amino acid sequence motif related to KFERQ[3,16].This pentapeptide target-ing motif wasfirst identified in microinjected RNase A[44].It consists of a glutamine(Q)preceded or followed bya combination of four amino acids that are basic(R,K), acidic(D,E),or bulky and hydrophobic(F,I,L,V)resi-dues.In some cases,Q may be substituted by the related N [45].Antibodies raised against KFERQ can immunopre-cipitate30%of cytosolic proteins in mammalian cells[46].752W.Li et al.Without denaturation,more than80%of cytosolic pro-teins that contain KFERQ motifs can be recognized by the antibody to this motif,indicating that most KFERQ motifs in these proteins are exposed.However,certain proteins such as aldolase B have hidden KFERQ motifs due to multimeric formation.The may dissociate the aldolase tetramer and result in the exposure of KFERQ motif[25,47].In addition,certain monomers may have hidden KFERQ sequences that may be exposed following partial unfolding.Therefore,the presence of a KFERQ sequence alone in the primary structure of a protein is not sufficient for determining them as substrates of CMA[48]. Such identification requires rigorous experimental proof. Recently,we have identified MEF2D,a protein known to promote neuronal survival as a CMA substrate.Interest-ingly,MEF2D uses a set of over-lapping imperfect KFERQ motifs to mediate its interaction with Hsc70[49]. Characteristics of CMACompared to macro-and microautophagy,which occurs in a wide range of eukaryotes including mammals,plants and fungi,CMA has only been described in mammals.This process has some distinctive features.SelectivityLysosome-mediated degradation had traditionally been perceived as a process performed in bulk with poor selectivity.The initial idea of selective autophagy origi-nated from the observation that starvation in animals or serum removal in cultured cells accelerates the degradation of particular cytosolic proteins in lysosomes but not others[50].Selectivity thus became one of the hallmarks of CMA[51].CMA mediates selective targeting of non-essential proteins for degradation to obtain the amino acids required for the synthesis of essential proteins.The intrinsic selec-tivity of CMA is also well suited for the removal of specific proteins damaged during stress without interfering with nearby normally functioning forms of the same protein[51, 52].This selectivity is achieved by making the KFERQ motifs in the altered protein accessible to the chaperone but inaccessible when it is properly folded or pare to CMA,macroautophagy has traditionally been consid-ered as a nonselective degradation process.However, recent evidence suggests that this view needs modification. New experimental data demonstrate that some macro-autophagic processes,termed chaperone-assisted selective autophagy,are assisted by chaperone proteins and can be selective in targeting protein complexes,organelles and microbes[2].In this process,macroautophagy has been proposed to be initiated by a selective ubiquitylation of cellular targets and followed by recognition via autophagic ubiquitin adaptors such as p62,NBR1and HDAC6.These molecules can mediate docking of ubiquitinated proteins or damaged organelles to autophagosomes and lysosomes, thereby ensuring their selective degradation[2,53,54].SaturabilityThe unusual characteristics of CMA are not limited to its selectivity.As the mechanism for cargo delivery was revealed,it became evident that,in contrast to the other forms of autophagy,vesicle formation was not required in CMA.Instead,the substrate proteins were translocated across the lysosomal membrane[55].The receptor Lamp2a is mainly responsible for this translocation.Because of the requirement for receptor binding before translocation can occur,this delivery process becomes saturable.In contrast to Hsc70,which is often in excess in the cytosol,levels of Lamp2a are limiting for CMA and hence subjected to tight regulation[23].CompetitivityThe selectivity and saturability of CMA directly lead to the competitive binding of CMA substrates.During the process of CMA,different substrates compete for binding Hsc70 and limited pool of Lamp2a[23,56].Several studies have shown that degradation of some substrates is slowed down by overexpression of other substrates.This has been proposed to underlie the pathogenesis of some diseases [49,57].For example,both the wild-type a-synuclein and neuronal transcription factor MEF2D are substrates of CMA.Elevated levels of wild-type a-synuclein may reduce the degradation of MEF2D via CMA[49].Regulation of CMAThe signal transduction pathways involved in the regula-tion of CMA from cell membranes to cytosolic chaperone proteins and lysosomes remain largely elusive[3].The p38 MAPK inhibitor can partly prevent the activation of CMA, implicating this pathway in CMA[45].On the other hand, the extensively investigated local regulation of CMA activity in the lysosomes has precise,fine-tuned mecha-nisms[3,25].Regulation of CMA via Lamp2aThe level of Lamp2a at the lysosomal membrane is pro-portional to the activity of CMA.Therefore,changes in the Lamp2a level at the lysosomal membrane can quickly regulate the activity of CMA.The level of Lamp2a at theBasics of chaperone-mediated autophagy753lysosomal membrane may be changed by synthesis,deg-radation and redistribution[41,58].De novo synthesis of Lamp2a or its by protein synthesis inhibitors can directly change the level of Lamp2a in cells,but the physiological or pathological relevance remains unclear [3,59].The distribution of Lamp2a at the lysosomal membrane is regulated by its dynamic association with discrete membrane lipid microdomains[60].Under basal conditions,sequestration of Lamp2a in cholesterol-enri-ched regions favors its cleavage by two proteases, including an unidentified metalloprotease at the membrane and a serine protease—cathepsin A,which associates dynamically with the lumenal side of the lysosomal mem-brane[60,61].Exclusion of Lamp2a from these regions allows its multimerization(see below),a step required for the uptake of CMA substrate by lysosomes.Interestingly, the degradation of Lamp2a can be reduced by the presence of substrates or stimuli that induce the activation of CMA. This blockage in degradation,rather than the increase of synthesis,to increase Lamp2a at the lysosomal membrane is particularly advantageous to cells with limited access to amino acids such as during nutrient deficit[3,62].The levels of Lamp2a can be further increased at the lysosomal membrane through the mobilization of the pool normally resident in the lysosomal lumen.The exact nature of this luminal pool of Lamp2a remains unclear,but intact molecules of this protein exist inside lysosomes,and a gradual decrease in the percentage of Lamp2in this com-partment occurs as activation of CMA persists beyond1 day[3,60].Membrane chaperones and an intact membrane potential are needed for the mobilization of Lamp2a from the lumen to membrane[3].Fractionation studies have shown that luminal Lamp2a associates with lipid,indicat-ing the possible existence of luminal Lamp2a-containing micelles.It is thought that these micelles fuse or integrate into the lysosomal membrane under specific stress,result-ing in the incorporation of Lamp2a in the membrane and exposure of its C terminus to the cytosol[60,63].Lamp2a can undergo cycles of rapid assembly into a 700-kDa protein complex at the lysosomal membrane. Monomers of Lamp2a at the lysosomal membrane can accept substrate proteins,and this interaction drives the organization of Lamp2a into multimeric complexes needed for substrate translocation into lysosomes.Once the sub-strate protein reaches the lysosomal lumen,Lamp2a will disassemble from the multimeric complex to enable sub-sequent rounds of substrate binding[3,40].This continuous assembly and disassembly of Lamp2a from the multimeric translocation complex highlights the impor-tance of the lateral mobility of this protein in the lysosomal membrane[40].Chaperone proteins located at both sides of the lysosomal membrane may regulate the lateral mobility of Lamp2a.Lys-Hsc70induces disassembly of Lamp2a from the700-kDa complex once the substrate has crossed the membrane.A lysosome-associated form of the glial fibrillary acidic protein(GFAP),a component of the intermediatefilament network,associates to Lamp2a once it is organized into multimers and contributes to stabilizing the CMA translocation complex against the disassembling activity of Hsc70,whereas GTP-mediated release of elongation factor-1a from the lysosomal membrane pro-motes self-association of GFAP,disassembly of the CMA translocation complex and the consequent decrease in CMA[12,64].In addition,Hsp90at the lumenal side is also required to preserve the stability of Lamp2a during the transition[40].Regulation of CMA via Hsc70The other limiting lysosomal component is lys-Hsc70.As indicated in the previous section,the presence of this chaperone at the luminal side of the membrane is necessary for substrate translocation[18].Levels of lys-Hsc70 increase gradually with increasing CMA activity,although the mechanisms modulating this increase are still poorly understood.Although Hsc70contains two KFERQ sequences and is a putative substrate of CMA[21,25],it appears that neither CMA nor macroautophagy is involved in delivering lys-Hsc70to lysosomes[3,65].It is possible that this chaperone reaches lysosomes through maturation of late endosomes,a compartment in which high levels of luminal Hsc70have been detected,thus highlighting a possible relationship between CMA and endocytosis [3,65,66].Regulation of CMA via macroautophagyThe activity of CMA is also directly modulated by changes in other autophagic and proteolytic systems inside the cell. Cells in culture respond to CMA blockage by upregulating macroautophagy.Similarly,blockage of macroautophagy results in constitutive activation of CMA[65].These pathways are clearly not redundant,as CMA is,for example,unable to degrade organelles normally turned over by macroautophagy,whereas macroautophagy lacks the selectivity of CMA in the degradation of individual soluble cytosolic proteins[67].Nevertheless,the compen-satory activation of one form of autophagy when the other is compromised allows cells to preserve homeostasis,at least under basal conditions.Additionally,blockage of either form of autophagy also has a direct impact on pro-teasomal activity[3].The molecular mechanisms that regulate crosstalk between these two different pathways are currently under investigation.In the case of the interrela-tionship between macroautophagy and CMA,continuous fusion of autophagosomes to lysosomes when754W.Li et al.macroautophagy is upregulated results in transient dissi-pation of the lumenal lysosomal pH,which negatively affects lys-Hsc70stability.In fact,although lys-Hsc70is normally stable at pH ranges of5.2–5.4,changes in pH values above5.6in the lysosomal lumen result in its rapid degradation in this compartment.The reduced levels of lys-Hsc70in those lysosomes decrease their capability to perform CMA[68].Regulation of CMA via UPSUPS and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. Increasing evidence shows that the UPS and autophagy are functionally interrelated catabolic processes.Specifi-cally,these degradation systems share certain substrates and regulatory molecules,and show coordinated and,in some contexts,compensatory function.For example,the neuronal protein a-synuclein can be degraded by the UPS,macroautophagy and CMA.Under conditions in which the UPS is compromised,enhanced degradation by CMA and macroautophagy may become critical to maintaining pools of amino acids for protein synthesis and may protect against the accumulation of a toxic species[1,3].On the other hand,during the acute stages of CMA blockage,there is an accumulation of poly-ubiquitinated proteins,often in the form of protein aggregates,attributable to the observed reduction in their removal through the proteasome system,and the under-lying mechanisms remain to be clarified[69]. Interestingly,recent evidence suggests that ubiquitin may play a key role in the crosstalk between proteasome-mediated degradation and selective autophagy[2,70]. Furthermore,ubiquilin,a ubiquitin-like protein,functions to regulate macroautophagy by facilitating maturation of LC3protein,and is also a substrate of CMA,indicating that ubiquilin may also be at a crossroad between protein degradation pathways[71,72].However,detailed mechanisms are under investigation.Physiological relevanceSelective degradation of cytosolic proteins via CMA con-tributes to both quality control(housekeeping)and response to stress[2,3].CMA was initially identified as an inducible pathway in response to stress.However, increasing evidence shows that there is a certain level of CMA activity under basal conditions.Most cell types analyzed to date display some level of continuous CMA activity detectable in the absence of typical CMA-inducing conditions.Basal CMA requires participation of the same effectors at the lysosomal membrane—the membrane chaperones and the protein translocation complex.It has been speculated that there is a difference between basal and inducible CMA.Whether the regulation of basal and inducible CMA occurs through different signaling mecha-nisms is under investigation[65].However,both basal and inducible CMA may have physiological relevance.Recycling and quality controlThe delivery of intracellular substrates such as misfolded proteins and damaged organelles from the cytosol to the lysosomes for degradation is crucial for cell survival. Under physiological conditions,renewal of cytosolic pro-teins is needed to maintain their normal function through recycling their old versions[4].Besides bulk autophagy (microautophagy and macroautophagy),CMA efficiently facilitates the transit of specific proteins from the cyto-plasm to lysosomes and is responsible for their recycling.If this process is inhibited,excessive old and dysfunctional proteins will accumulate in the cytosol and disturb the physiological functions of cells[3,49].Immune responseDurable adaptive immunity is dependent on CD4?T cell recognition of the major histocompatibility complex (MHC)class II molecules that display peptides from exogenous and endogenous antigens.Specialized antigen-presenting cells use the endosomal/lysosomal systems to internalize exogenous antigens,which can then be pre-sented on MHC to CD4?T cells.Beside the proteasome and macroautophagy in processing and MHC loading with endogenous and exogenous antigens,CMA has also been recently investigated in antigen processing/presentation. Cells with reduced levels of Lamp2a or Hsc70exhibit decreased presentation of cytoplasmic epitopes on class II molecules[73].Conversely,an increase in cytoplasmic autoantigen presentation is observed upon overexpression of either Lamp2a or Hsc70.Furthermore,there is cross-talk between autophagy pathways in the expression of MHC class II molecules.Macroautophagy can deliver antigens into autophagosomes for processing by acidic proteases before MHC class II presentation.However,other endog-enous antigens are processed by cytoplasmic proteases, yielding fragments that translocate via CMA into the endosomal network to intersect MHC class II.This cross-talk,particularly in response to stress,appears to balance the relative efficiency of each pathway,limits redundancy and gives MHC class II broader access to antigens within different intracellular compartments.Whether alteration in CMA activity such as its reduction with age could con-tribute to the altered immune response requires further investigation[74].Basics of chaperone-mediated autophagy755。

84猪业科学  SWINE INDUSTRY SCIENCE 2013年 第12期猪轮状病毒属于呼肠孤病毒科轮状病毒属，是引起多种新生动物和幼龄动物腹泻的重要肠道病原之一，也是感染1～5日龄仔猪或断奶仔猪的常见病原。

以厌食、呕吐、腹泻和脱水、体重减轻为特征，引起仔猪的死亡，成年猪掉膘，饲料报酬降低，增加人工费和药费的开支等。

若与传染性胃肠炎和大肠杆菌混和感染则病情加重,死亡率增高。

1 猪轮状病毒形态成熟完整轮状病毒粒子略呈圆形，没有囊膜，为65～75 nm 的二十面体对称。

电镜观察，病毒的中央为核酸构成的芯髓，呈致密的六角形棱心，直径为37～40 nm。

轮状病毒粒子由3层衣壳组成：内衣壳（VP2）、中间层衣壳(VP6)和外层衣壳（VP4和VP7）。

中间层衣壳(VP6)有32个呈放射状排列的圆柱形壳粒组成，壳粒由此向外呈辐射状排列。

外衣壳厚约20 nm，为连接于壳粒末端的光滑薄膜状结构，使该病毒形成车轮状外观，轮状病毒为此而得名（见图1、图2）。

在感染的粪样和细胞培养物中存在2种形式的病毒粒子，只有3层衣壳的光滑型（S 型）粒子，具有感染性[1]，直径为75 nm。

外层衣壳被除去，就会导致感染性丧失，表明钙离子通过使外层衣壳VP7稳定，对轮状病毒粒子的稳定性起着关键作用[2]。

不同类型的病毒，稳定外层衣壳所用钙的浓度不一样[3]。

没有外层衣壳的双层粒子（R 型）是很小的，边缘粗糙，直径为65 nm。

病毒核酸基因由11 个不连续的 猪轮状病毒病原学研究进展王振玲1，李显和2（1.北京农业职业学院，北京 房山 102442；2.北京市大兴区魏善庄镇动物防疫畜牧水产技术推广站，北京 大兴 102611）摘 要：轮状病毒不仅感染猪、牛、羊、禽等动物，同时还感染人。

轮状病毒的持续存在，不仅严重威胁养殖业的发展，同时威胁到肉食品和人类的安全，成为世界科学家们研究的焦点。

对轮状病毒形态、分类、理化性质和生物学特性、致病机理等病原学等内容进行了综述。

热休克蛋白70与心肌保护研究现状（作者:___________单位: ___________邮编: ___________）【关键词】热休克蛋白质70 心肌缺血心肌再灌注损伤/治疗热休克蛋白(heat shock protein, HSP)是指细胞在应激原特别是环境高温诱导下所产生的，由热休克基因所编码合成的，序列高度保守的一组蛋白质。

近年来研究发现，以热、缺血缺氧为应激原或通过基因调控的方法诱导心肌细胞产生热休克蛋白，可使心肌具有抗热、抗缺血缺氧的保护作用。

热休克蛋白按其分子量不同分为多个家族，其中，热休克蛋白70(HSP70)备受重视，已成为新近较重视的内源性心肌保护途径之一，在对抗心肌缺血再灌注损伤中发挥着极为重要的作用。

综合近几年的研究热点，现就其特性及其在心肌保护方面的研究现状进行综述。

1 HSP的一般概况1962年Ritossa［1］首次从果蝇在热应激环境中诱导合成而命名，随后的研究发现，除高温外，缺血、低氧、重金属盐及大多数病理状态下都能诱导细胞产生热休克蛋白，因此又称应激蛋白，但习惯上仍沿用热休克蛋白这一名称。

根据分子量大小和同源程度可分为HSP110，HSP90，HSP70，HSP60及小分子量HSP等几个家族，每个家族又有多个成员。

HSP70是HSP家族中最保守、最主要、含量最丰富，也是近年来研究备受关注的一类。

HSP70家族(分子量：72～80)成员最多，共有21种蛋白质，是一组进化上高度保守的应激蛋白。

按表达情况可分为结构型HSP70和诱导型HSP70，生理情况下，正常细胞可表达结构型HSP70，应激情况下略有增加。

结构型HSP70又可分为两种，即葡萄糖调节蛋白78(glucose-regulated protein, GRP78)，这种蛋白存在于内质网腔内；另一种是GRP75，主要存在于线粒体内。

诱导型HSP70即HSP72，仅出现于应激细胞，通常在正常细胞中并不表达或表达量甚少，但是在热应激或其它应激原的作用下则表达迅速增加［2］。

水稻S5—ORF3蛋白质的进化分析作者：张达瀚张屹来源：《河北科技大学学报》2014年第04期摘要：生殖隔离在形成新物种和物种同一性的保持中有重要作用，因而在水稻优化育种中有重要的意义。

S5位点是一个已克隆的控制水稻生殖隔离的基因，它产生的S5-ORF3，ORF4和ORF5蛋白质共同调控着indica-japonica杂交后代的育性，特别是S5-ORF3蛋白质在打破水稻亚种indica-japonica之间的生殖隔离与促进物种间的基因交流中发挥重要作用。

针对S5-ORF3的进化分析对于研究它的功能和起源很重要，但目前尚无报道。

本文基于序列和进化分析，提出S5-ORF3为一种定位在内质网中的新的HSP70家族蛋白，但缺乏HSP70的C端的多肽结合结构域，推测S5-ORF3可能通过影响alpha-淀粉酶的合成来作用于内质网压力；找到了ORF3的19条同源蛋白，并用极大似然算法构建了可靠的进化树，发现水稻的S5-ORF3与节节麦的luminal-binding蛋白进化关系最为密切；作了置信度100%的S5-ORF3蛋白质的三维结构预测，预测了居中的配体绑定位点；发现了水稻的S5-ORF3与其他HSP70蛋白相比独特的基序，为进一步研究S5-ORF3的作用机理和演化历史提供了线索和数据支持。

关键词：水稻；S5-ORF3 蛋白；进化树；正向选择；配体结合中图分类号：Q816 文献标志码：A生殖隔离限制了物种间的基因交流，一般来说，水稻的2个亚种indica和 japonica间产生的后代是不育的[1-2]。

研究发现，在水稻基因组的S5位点有一个与生殖隔离相关的“破坏与保护蛋白系统”，该系统由3个紧密连锁的基因ORF3，ORF4，ORF5组成，这三者共同调控着indica-japonica杂交后代的育性，ORF4+与ORF5+导致了内质网的压力，这种压力导致杂交水稻雌配子败育，而ORF3+作为保护者又会阻止内质网压力的产生，从而抑制雌配子败育并打破生殖隔离，使indica和japonica之间的基因可以交流[3]。

收稿日期:1999-10-20;修订日期:2000-12-30基金项目: 国家自然科学研究基金资助(39770824)作者简介:范云霞(1964-),女(汉族),河南平顶山市,中国协和医科大学生物化学硕士研究生审校者:中国协和医科大学肿瘤研究所 黄常志026 热休克蛋白与免疫应答范云霞(中国协和医科大学肿瘤研究所,北京 100021))摘要:近年研究发现,热休克蛋白参与免疫应答过程,在抗原受体成熟、抗原加工、呈递等多方面起作用,并且这种作用具有潜在的临床应用前景,本文综述这一领域的研究现状。

关键词:热休克蛋白;免疫应答;抗原受体;抗原加工呈递文章编号:1001-103X(2001)02-0062-03 中图分类-号:R392 11 文献标识码:A 热休克蛋白(Heat Shock Protein,HSP)是一组具有重要生理功能,高度保守的蛋白质分子家族。

生理、病理及环境因素等都可诱导热休克蛋白产生,故又称为应激蛋白(Stress Protein)。

根据分子量大小和同源程度可分为HSP110、HSP90、HSP70、HSP60、小分子HSP 及泛素等几个家族。

热休克蛋白的生物学功能广泛,不仅表现在应激条件下维持细胞必需的蛋白质空间构象,保护细胞生命活动,以确保细胞生存,而且在蛋白质折叠、跨膜运输、转位、细胞骨架及核骨架稳定等基本功能方面发挥重要作用,调节这些蛋白的活性和功能。

而自身并不参与大分子蛋白组成,故称为 分子伴侣 。

本文主要综述HSP70、HSP90、泛素等在抗原受体装配、抗原加工与呈递等方面的作用。

1 HSP70、HSP90、泛素组成和结构HSP70家族成员广泛存在细胞的各个亚细胞结构、胞浆及胞核中,如:组成性表达HSC70(Heat Shock Cognate Protein,HSC70),热诱导表达HSP70,定位于内体和质膜PBP72/74(Peptide Binding Protein,PBP),线粒体基质中GRP75(Glucose Regulated Protein,GRP),内质网腔丰富存在BiP(Immunoglobulin hea vy chain binding Protein,BiP)。

HSP70的研究进展及其在生物医学中的应用作者：刘嘉敏赵元莙来源：《教育教学论坛》2017年第50期摘要：HSP70是热休克蛋白家族的重要组成成员，均属机体内的一类应激蛋白，具有保护细胞和生命体的重要作用。

本文从HSP70家族各成员的研究概况、生物学功能及其在生物医学方面的应用予以简要综述，以期为中小学的素质教育提供生物医学方面的普及知识。

关键词：热休克蛋白；分类；功能；生物医学；应用中图分类号：G642.0 文献标志码：A 文章编号：1674-9324（2017）50-0061-02热休克蛋白（HSPs）是指在机体遇高温刺激及其他一些环境压力，如营养缺乏、氧压、重金属、强紫外线照射及细菌等生物污染有害刺激后，诱导机体所产生的高度保守的、可帮助细胞耐受这些环境压力并对机体起着重要防御作用的一组蛋白质。

依据分子量大小，热休克蛋白可分为HSP100、HSP90、HSP70、HSP60、HSP40、小HSP家族和泛素等家族。

目前，热休克蛋白70（HSP70）是热休克家族（HSPs）中研究最多的应激蛋白，该类蛋白在原核生物和真核生物中广泛存在。

一、HSP70的分类及功能在生物细胞中，HSP70含量丰富且分布广泛，它们不仅分布于细胞质中，有些还存在于细胞器中。

HSP70 家族主要包括四类蛋白质：（1）诱导型 HSP70（也称HSP72），存在于细胞核中，在正常细胞内表达量较少，细胞发生应激后，表达量迅速增加，与ATP具有高亲和力。

（2）结构型HSC70（Heat shock cognate 70，也称HSP 73），存在于细胞浆中，在所有的细胞内组成性表达，是哺乳动物细胞内的结构蛋白，外界应激原刺激后只有少量增加，与ATP 也具有高亲和力。

（3）葡萄糖调节蛋白78（GRP78）（也称HSPA5）位于内质网上，应激条件下稍有表达，参与多种细胞生命过程。

（4）葡萄糖调节蛋白75（HSP75）（也称HSPA9），主要位于线粒体内，其基因能够在多种组织中表达，且表达量和细胞自身的能量代谢有关。

收稿日期：2013-11-05基金项目：国家自然科学基金项目(81041088) *通信作者：E-mail: hchen@ 细胞内吞的研究进展范真真，陈虹*，黄秉仁(中国医学科学院&北京协和医学院基础医学研究所医学分子生物学国家重点实验室，北京 100005)摘要：细胞外基质的各种分子经细胞膜进入真核细胞是一个复杂的过程。

细胞内吞是通过细胞质膜的变形运动将细胞外物质转运入细胞内的过程。

不同的细胞内吞途径需要不同的蛋白质分子参与，引起不同的信号转导通路。

目前认为细胞内吞和膜转运是细胞对其信号转导过程的一种精密的组织安排，细胞内吞在细胞信号转导，维持机体动态平衡方面起着重要作用。

细胞内吞途径通常可以分为网格蛋白依赖的内吞和非网格蛋白依赖的内吞，其中后者包括陷窝蛋白依赖和非陷窝蛋白依赖的内吞，以及巨胞饮介导的内吞。

本文将就这几种主要细胞内吞途径及与细胞信号转导通路关系的研究进展予以介绍。

关键词：细胞内吞；网格蛋白；陷窝蛋白；发动蛋白；巨胞饮Research progress of cell endocytosisFAN Zhenzhen, CHEN Hong*, HUANG Bingren(The National laboratory of Medical Molecular Biology, Department of Biochemistry & Molecular Biology,Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine,Peking Union Medical College, Beijing 100005, China)Abstract: It is a complex process for the movement of multiple extracellular matrix molecules crossing the eukaryotic cell membrane. Endocytosis is such a process, through which extracellular materials can be transported across the membrane. Different endocytosis pathways require the participation of diverse protein factors, which can activate different signaling pathways. Currently, endocytosis and membrane trafficking are considered to be precise and highly organized in cellular signaling transduction system. In addition, endocytosis plays key roles in cellular signaling and homeostasis. In general, endocytosis can be divided into two types: the clathrin-dependent and clathrin-independent endocytosis pathways. The clathrin-independent endocytosis pathways could be subdivided into three categories: caveolin-dependent endocytosis, caveolin-independent endocytosis and macropinocytosis. In this review, we will outline the research advance in the relationship be-tween the main endocytosis pathways and cell signaling transduction.Key Words: cell endocytosis; clathrin; caveolin; dynamin; macropinocytosis细胞内吞作用(endocytosis)长期以来一直被认为是细胞将营养物质和与膜相关分子内置化的一个简单过程。

生物技术进展２０１９年㊀第９卷㊀第４期㊀３３２~３４０ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ㊀ＩＳＳＮ２０９５￣２３４１进展评述Ｒｅｖｉｅｗｓ㊀收稿日期:２０１９￣０３￣１８ꎻ接受日期:２０１９￣０４￣２４㊀作者简介:闫东科ꎬ助教ꎬ研究方向为生物化学与分子生物学ꎮＥ￣ｍａｉｌ:９８３０６９５２５＠ｑｑ.ｃｏｍꎮ∗通信作者:闫东科与吕平为共同通信作者ꎮ吕平ꎬ教授ꎬ研究方向为生物技术ꎮＥ￣ｍａｉｌ:Ｂｅｓｔｍａｎ＿０４２９＠１６３.ｃｏｍ低氧诱导因子及其抑制剂研究进展闫东科∗ꎬ㊀吕㊀平∗天津职业大学生物与环境工程学院食品生物技术系ꎬ天津３００３５０摘㊀要:低氧(ｈｙｐｏｘｉａ)是生物体内常见的生理和病理现象ꎬ也是常见的实体肿瘤微环境ꎮ人和其他哺乳动物体内存在一系列应对缺氧胁迫的调节机制ꎬ而低氧诱导因子(ｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓꎬＨＩＦｓ)是这些调节机制中的关键调控因子ꎬ是一类参与细胞缺氧应答反应的转录因子家族ꎮ大量研究表明ꎬＨＩＦｓ与生物体的生长㊁发育㊁血管生成㊁红细胞生成㊁细胞代谢和自噬以及肿瘤的发生㊁发展㊁转移侵袭㊁放化疗抗性和癌症患者的不良预后等密切相关ꎮ因此ꎬ进一步加强ＨＩＦｓ及其抑制剂的相关研究对于肿瘤的认识与治疗具有重要意义ꎮ从ＨＩＦｓ的蛋白质结构㊁稳定性调控㊁转录激活调控及其靶向抑制剂的研究进展等方面进行综述ꎬ以期为靶向ＨＩＦｓ的肿瘤治疗提供新线索ꎬ为新型ＨＩＦｓ抑制剂的研究与开发提供参考ꎮ关键词:低氧诱导因子ꎻ稳定性调控ꎻ转录活性ꎻ靶向抑制剂ꎻ肿瘤治疗ＤＯＩ:１０.１９５８６/ｊ.２０９５￣２３４１.２０１９.００２７ＰｒｏｇｒｅｓｓｏｎＨｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅＦａｃｔｏｒａｎｄｉｔｓＩｎｈｉｂｉｔｏｒｓＹＡＮＤｏｎｇｋｅ∗ꎬＬＹＵＰｉｎｇ∗ＤｅｐａｒｔｍｅｎｔｏｆＦｏｏｄＢｉｏｔｅｃｈｎｏｌｏｇｙꎬＳｃｈｏｏｌｏｆＢｉｏｌｏｇｉｃａｌａｎｄＥｎｖｉｒｏｎｍｅｎｔａｌＥｎｇｉｎｅｅｒｉｎｇꎬＴｉａｎｊｉｎＶｏｃａｔｉｏｎａｌＩｎｓｔｉｔｕｔｅꎬＴｉａｎｊｉｎ３００３５０ꎬＣｈｉｎａＡｂｓｔｒａｃｔ:Ｈｙｐｏｘｉａｉｓａｃｏｍｍｏｎｐｈｙｓｉｏｌｏｇｉｃａｌａｎｄｐａｔｈｏｌｏｇｉｃａｌｐｈｅｎｏｍｅｎｏｎｉｎｖｉｖｏａｎｄａｃｏｍｍｏｎｍｉｃｒｏｅｎｖｉｒｏｎｍｅｎｔｏｆｓｏｌｉｄｔｕｍｏｒｓ.Ｔｈｅｒｅａｒｅａｓｅｒｉｅｓｏｆｒｅｇｕｌａｔｉｏｎｍｅｃｈａｎｉｓｍｓｉｎｈｕｍａｎｓａｎｄｏｔｈｅｒｍａｍｍａｌｓｔｏｄｅａｌｗｉｔｈｈｙｐｏｘｉａｓｔｒｅｓｓ.Ａｎｄｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓ(ＨＩＦｓ)ａｒｅｔｈｅｋｅｙｒｅｇｕｌａｔｏｒｓｏｆｔｈｅｓｅｒｅｇｕｌａｔｉｏｎｍｅｃｈａｎｉｓｍｓꎬａｎｄｔｈｅｙａｒｅａｆａｍｉｌｙｏｆｔｒａｎｓｃｒｉｐｔｉｏｎｆａｃｔｏｒｓｉｎｖｏｌｖｅｄｉｎｃｅｌｌｕｌａｒｈｙｐｏｘｉａｒｅｓｐｏｎｓｅ.ＥｘｔｅｎｓｉｖｅｓｔｕｄｉｅｓｈａｖｅｓｈｏｗｎｔｈａｔＨＩＦｓａｒｅｃｌｏｓｅｌｙｒｅｌａｔｅｄｔｏｇｒｏｗｔｈａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆｏｒｇａｎｉｓｍｓꎬａｎｇｉｏｇｅｎｅｓｉｓꎬｅｒｙｔｈｒｏｐｏｉｅｓｉｓꎬｃｅｌｌｍｅｔａｂｏｌｉｓｍａｎｄａｕｔｏｐｈａｇｙꎬａｎｄｏｃｃｕｒｒｅｎｃｅꎬｄｅｖｅｌｏｐｍｅｎｔꎬｍｅｔａｓｔａｓｉｓａｎｄｉｎｖａｓｉｏｎꎬｒａｄｉｏｃｈｅｍｏｔｈｅｒａｐｙｒｅｓｉｓｔａｎｃｅｏｆｔｕｍｏｒａｓｗｅｌｌａｓｐｏｏｒｐｒｏｇｎｏｓｉｓｏｆｃａｎｃｅｒｐａｔｉｅｎｔｓ.ＴｈｅｒｅｆｏｒｅꎬｉｔｉｓｏｆｇｒｅａｔｓｉｇｎｉｆｉｃａｎｃｅｔｏｆｕｒｔｈｅｒｓｔｒｅｎｇｔｈｅｎｔｈｅｒｅｓｅａｒｃｈｏｎＨＩＦｓａｎｄｔｈｅｉｒｉｎｈｉｂｉｔｏｒｓｆｏｒｔｈｅｕｎｄｅｒｓｔａｎｄｉｎｇａｎｄｔｒｅａｔｍｅｎｔｏｆｔｕｍｏｒｓ.ＴｈｅｐｒｏｇｒｅｓｓｏｆｐｒｏｔｅｉｎｓｔｒｕｃｔｕｒｅꎬｓｔａｂｉｌｉｔｙｒｅｇｕｌａｔｉｏｎꎬｔｒａｎｓｃｒｉｐｔｉｏｎａｌａｃｔｉｖａｔｉｏｎｒｅｇｕｌａｔｉｏｎａｎｄｔａｒｇｅｔｅｄｉｎｈｉｂｉｔｏｒｓｏｆＨＩＦｓｗａｓｒｅｖｉｅｗｅｄꎬｉｎｏｒｄｅｒｔｏｐｒｏｖｉｄｅｎｅｗｉｎｓｉｇｈｔｓｉｎＨＩＦｓｔａｒｇｅｔｅｄｔｕｍｏｒｔｈｅｒａｐｙａｎｄｏｆｆｅｒｒｅｆｅｒｅｎｃｅｓｆｏｒｔｈｅｒｅｓｅａｒｃｈａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆｎｅｗＨＩＦｓｉｎｈｉｂｉｔｏｒｓ.Ｋｅｙｗｏｒｄｓ:ＨＩＦｓꎻｓｔａｂｉｌｉｔｙｒｅｇｕｌａｔｉｏｎꎻｔｒａｎｓｃｒｉｐｔｉｏｎａｌａｃｔｉｖｉｔｙꎻｔａｒｇｅｔｅｄｉｎｈｉｂｉｔｏｒꎻｔｕｍｏｒｔｈｅｒａｐｙ㊀㊀低氧是生物体内常见的生理和病理现象ꎬ常发生于早期胚胎发育㊁肿瘤形成㊁急慢性血管疾病和慢性阻塞性肺病等ꎮ为了应对低氧胁迫ꎬ生物体内存在一系列调节机制ꎬ而在这些调节途径中ꎬＨＩＦｓ是最主要的一类介导低氧适应性应答反应的转录因子家族ꎮＨＩＦｓ调控的靶基因产物涉及红细胞生成㊁血管生成以及细胞的增殖㊁代谢和凋亡等多个方面ꎬ因而在人和哺乳动物的低氧适应生理性应答反应中发挥着重要作用ꎻ而ＨＩＦｓ在机体的低氧适应病理性应答反应中的作用更值得关注ꎬ如ＨＩＦｓ可调控肿瘤细胞的代谢重编程㊁抑制肿瘤细胞凋亡㊁诱导自噬促进肿瘤细胞存活ꎬ并与新生血管生成㊁上皮间质转化(ｅｐｉｔｈｅｌｉａｌ￣ｍｅｓｅｎｃｈｙｍａｌｔｒａｎｓｉｔｉｏｎꎬＥＭＴ)㊁转移侵袭㊁放化疗抗性㊁ｐＨ稳态㊁自分泌㊁肿瘤干细胞(ｔｕｍｏｒｓｔｅｍｃｅｌｌꎬＣＳＣ)维持和肿瘤预后等密切相关ꎬ从而起到促进肿瘤发生发展的作用[１~４]ꎮ因此ꎬ加强ＨＩＦｓ生物学功能与特性及其. All Rights Reserved.作用调控机制的研究ꎬ是开发新型抗癌药物的关键ꎮ本文将从ＨＩＦｓ的蛋白质结构㊁稳定性调控㊁转录激活调控及其靶向抑制剂的研究进展等方面进行综述ꎬ以期为靶向ＨＩＦｓ的肿瘤治疗提供新线索ꎬ为新型ＨＩＦｓ抑制剂的研究与开发提供参考ꎮ１㊀ＨＩＦｓ蛋白质结构人和其他哺乳动物体内共存在３种ＨＩＦｓ家族成员ꎬ即ＨＩＦ￣１㊁ＨＩＦ￣２和ＨＩＦ￣３ꎬ三者均为由受氧气浓度调控的ＨＩＦ￣α亚基(ＨＩＦ￣１α㊁ＨＩＦ￣２α和ＨＩＦ￣３α)和组成型表达的ＨＩＦ￣１β亚基(又称芳香烃受体核转运蛋白ꎬａｒｙｌｈｙｄｒｏｃａｒｂｏｎｒｅｃｅｐｔｏｒｎｕｃｌｅａｒｔｒａｎｓｌｏｃａｔｏｒꎬＡＲＮＴ)组成的异源二聚体ꎮ３种ＨＩＦ￣α亚基(ＨＩＦ￣αｓ)和ＨＩＦ￣１β亚基的Ｎ端均含有碱性螺旋－环－螺旋(ｂａｓｉｃｈｅｌｉｘ￣ｌｏｏｐ￣ｈｅｌｉｘꎬｂＨＬＨ)结构域㊁ＰＡＳ(Ｐｅｒ/ＡＲＮＴ/Ｓｉｍ)￣Ａ和ＰＡＳ￣Ｂ结构域(图１)ꎬ其中ꎬｂＨＬＨ￣ＰＡＳ结构域介导ＨＩＦｓ的异源二聚化以及ＨＩＦｓ与靶基因增强子或启动子上的低氧应答元件(ｈｙｐｏｘｉａｒｅｓｐｏｎｓｅｅｌｅ￣ｍｅｎｔꎬＨＲＥꎬ５ᶄ￣Ａ/ＧＣＧＴＧ￣３ᶄ)结合[５]ꎮＨＩＦ￣１α和ＨＩＦ￣２α亚基的Ｃ端含有氧依赖的降解结构域(ｏｘｙｇｅｎ￣ｄｅｐｅｎｄｅｎｔｄｅｇｒａｄａｔｉｏｎｄｏｍａｉｎꎬＯＤＤ)和２个转录激活结构域Ｎ￣ＴＡＤ和Ｃ￣ＴＡＤ(Ｎ/Ｃ￣ｔｅｒｍｉｎａｌａｃｔｉｖａｔｉｏｎｄｏｍａｉｎ)ꎮＨＩＦ￣３α亚基的Ｃ端含有ＯＤＤ结构域和Ｎ￣ＴＡＤ结构域ꎬ但缺少Ｃ￣ＴＡＤ结构域(图１)ꎮＯＤＤ结构域中的ＬＡＰＹＩＸＭＤ基序在ＨＩＦ￣αｓ与肿瘤抑制蛋白ＶＨＬ(ｖｏｎＨｉｐｐｅｌ￣ＬｉｎｄａｕｔｕｍｏｒｓｕｐｐｒｅｓｓｏｒｐｒｏｔｅｉｎꎬｐＶＨＬ)的结合中起关键作用[６]ꎻＮ￣ＴＡＤ结构域在ＨＩＦ￣αｓ的靶基因调控特异性中起主导作用ꎻＣ￣ＴＡＤ结构域具有富集辅助因子ｐ３００/ＣＢＰ形成转录激活复合物的作用[７]ꎮ此外ꎬＨＩＦ￣１α和ＨＩＦ￣２α亚基的Ｃ端存在双向核定位信号(ｎｕｃｌｅａｒｌｏｃａｌｉｚａｔｉｏｎｓｉｇｎａｌꎬＮＬＳ)ꎬ以确保二者定位于细胞核[８]ꎻＨＩＦ￣３α亚基的Ｃ端存在２段功能上冗余的ＮＬＳꎬ且第一段ＮＬＳ的核定位功能强于第二段[９]ꎮＨＩＦ￣３α亚基由于选择性剪接㊁转录启动子不同和翻译起始密码子位点不同等原因ꎬ存在多种剪接变异体[１０]ꎮ如人源ＨＩＦ￣３α(ｈＨＩＦ￣３α)有８种剪接变异体[１１]ꎬ其中全长的ｈＨＩＦ￣３α１含有亮氨酸拉链(ｌｅｕｃｉｎｅｚｉｐｐｅｒꎬＬＺＩＰ)基序和存在ＬＡ￣ＰＹＩＸＭＤ基序的ＯＤＤ结构域(图１)ꎻ而ｈＨＩＦ￣３α４剪接体仅含有ｂＨＬＨ￣ＰＡＳ结构域(图１)ꎬ且对ＨＩＦ￣１α和ＨＩＦ￣２α具有负调控作用[１２]ꎮＨＩＦｓ除了通过与靶基因上的ＨＲＥ直接结合激活转录ꎬ还可通过干扰其他转录因子(如Ｍｙｃ㊁ｐ５３和Ｎｏｔｃｈ等)的活性间接影响基因表达[１３]ꎮ如ＨＩＦ￣１和ＨＩＦ￣２竞争性地与Ｎｏｔｃｈ受体的胞内结构域(ＮｏｔｃｈｉｎｔｒａｃｅｌｌｕｌａｒｄｏｍａｉｎꎬＮＩＣＤ)相互作用ꎬ当ＨＩＦ￣２α与ＮＩＣＤ相互作用时ꎬ将抑制Ｎｏｔｃｈ信号通路的活性ꎬ而低氧对胶质瘤干细胞(ｇｌｉｏｍａｓｔｅｍｃｅｌｌｓꎬＧＳＣ)的增殖和自我更新等的促进作用是通过激活Ｎｏｔｃｈ信号通路实现的ꎻ与此相反ꎬ当ＨＩＦ￣１α与ＮＩＣＤ相互作用时ꎬ将活化Ｎｏｔｃｈ信号通路ꎬ增加低氧时Ｎｏｔｃｈ靶基因的表达ꎬ促进ＧＳＣ的增殖[１４]ꎮ２㊀ＨＩＦｓ的稳定性调控２.１㊀氧依赖稳定性调控机制ＨＩＦｓ作为细胞内氧动态平衡的感受器ꎬＨＩＦ￣αｓ亚基的稳定性受到氧浓度的严格监控ꎬ氧浓度图１㊀ＨＩＦ￣αｓ㊁ＨＩＦ￣１β㊁ｈＨＩＦ￣３α１和ｈＨＩＦ￣３α４的结构Ｆｉｇ.１㊀ＳｔｒｕｃｔｕｒｅｓｏｆＨＩＦ￣αｓꎬＨＩＦ￣１βꎬｈＨＩＦ￣３α１ａｎｄｈＨＩＦ￣３α４.注:ＩＤ:抑制结构域(ｉｎｈｉｂｉｔｏｔｙｄｏｍａｉｎ)ꎮ３３３闫东科ꎬ等:低氧诱导因子及其抑制剂研究进展. All Rights Reserved.的变化可引起ＨＩＦ￣αｓ亚基蛋白质水平的改变ꎬ这种氧依赖的稳定性调控机制以Ｏ２/ＰＨＤｓ/ｐＶＨＬ降解途径较为经典ꎮ常氧时ꎬ由ｐＶＨＬ㊁延伸蛋白ＥｌｏｎｇｉｎＢ和ＥｌｏｎｇｉｎＣ等组成的Ｅ３泛素连接酶复合物催化ＨＩＦ￣１/２α亚基中的Ｌｙｓ残基发生多聚泛素化修饰ꎬ而后由２６Ｓ蛋白酶体降解ꎬ其中ｐＶＨＬ直接与ＨＩＦ￣１/２α亚基结合ꎮ在多聚泛素化修饰发生前ꎬＨＩＦ￣１/２α亚基ＯＤＤ结构域中特异的Ｐｒｏ残基[１５] (如ＨＩＦ￣１α中的Ｐ４０２和Ｐ５６４残基ꎬＨＩＦ￣２α中的Ｐ４０５和Ｐ５３１残基)被以Ｏ２㊁α￣酮戊二酸为底物ꎬ以Ｆｅ２＋㊁抗坏血酸盐为辅酶的脯氨酸羟化酶(ｐｒｏｌｙｌｈｙｄｒｏｘｙｌａｓｅｄｏｍａｉｎｐｒｏｔｅｉｎｓꎬＰＨＤｓ)羟基化ꎮ羟基化修饰作用促进ＨＩＦ￣１/２α亚基与ｐＶＨＬ的结合ꎮ该降解途径称为Ｏ２/ＰＨＤｓ/ｐＶＨＬ途径(图２)ꎮＰＨＤ是该途径的关键限速酶ꎬ在哺乳动物体内存在４种ＰＨＤꎬ即ＰＨＤ１~４ꎬ其中ＰＨＤ２主要负责调节ＨＩＦ￣１α的降解ꎬＰＨＤ１和ＰＨＤ３主要负责调节ＨＩＦ￣２α的降解[１６]ꎮＤｕａｎ[６]在对脊椎动物中ＨＩＦ￣αｓ亚基的氨基酸序列同源性进行分析时发现ꎬｈＨＩＦ￣３α１中的Ｐ４０６/Ｐ４９２/Ｌ５０２残基和斑马鱼ＨＩＦ￣３α１中的Ｐ３９３/Ｐ４９３/Ｌ５０３残基均对应于ｈＨＩＦ￣１α亚基中被ＰＨＤ羟基化的氨基酸位点和与ｐＶＨＬ结合的氨基酸位点ꎮ上述氨基酸残基突变后可提高相应ＨＩＦ￣３α１蛋白的稳定性ꎮ推测全长ＨＩＦ￣３α亚基及包含有ＯＤＤ结构域的ＨＩＦ￣３α剪接体也可经Ｏ２/ＰＨＤｓ/ｐＶＨＬ途径降解ꎮ图２㊀ＨＩＦ￣α亚基的Ｏ２/ＰＨＤｓ/ｐＶＨＬ降解途径Ｆｉｇ.２㊀ＤｅｇｒａｄａｔｉｏｎｐａｔｈｗａｙｏｆＨＩＦ￣αｂｙＯ２/ＰＨＤｓ/ｐＶＨＬ.㊀㊀低氧时ꎬ细胞内琥珀酸㊁延胡索酸㊁活性氧(ｒｅａｃｔｉｖｅｏｘｙｇｅｎｓｐｅｃｉｅｓꎬＲＯＳ)等的积累以及ＣｏＣｌ２㊁二甲基乙二酰基甘氨酸(ｄｉｍｅｔｈｙｌｏｘａｌｙｌｇ￣ｌｙｃｉｎｅꎬＤＭＯＧ)㊁铁离子螯合剂等化学物质均可抑制ＰＨＤ的羟基化活性ꎬ从而阻断Ｏ２/ＰＨＤｓ/ｐＶＨＬ降解途径ꎬ使ＨＩＦｓ得以稳定ꎮ其中ꎬＤＭＯＧ为α￣酮戊二酸的结构类似物ꎬ是ＰＨＤ的竞争性抑制剂[１７]ꎻ而ＰＨＤ的催化中心含有Ｆｅ２＋ꎬ所以铁离子螯合剂也可抑制其活性ꎮ２.２㊀非氧依赖稳定性调控机制近年来的研究表明ꎬＨＩＦ￣α亚基的稳定性除受上述经典氧依赖降解途径的调控外ꎬ还受到非氧依赖机制的调控ꎮＨＩＦ￣αｓ亚基的非氧依赖稳定性调控机制以分子伴侣介导的自噬(ｃｈａｐｅｒｏｎｅ￣ｍｅｄｉａｔｅｄａｕｔｏｐｈ￣ａｇｙꎬＣＭＡ)使ＨＩＦ￣１α亚基在溶酶体中被降解为代表ꎮＣＭＡ是一种选择性自噬ꎬ负责降解胞质中近３０％的因氧化损伤的可溶性蛋白质ꎬ这些蛋白质均含有ＫＦＥＲＱ样五肽基序[１８]ꎮ在ＣＭＡ介导的溶酶体降解ＨＩＦ￣１α亚基的途径中ꎬ分子伴侣ＨＳＰＡ８/ＨＳＣ７０通过识别ＨＩＦ￣１α中的ＫＦＥＲＱ样基序与其结合ꎬ在使ＨＩＦ￣１α亚基肽链伸展后将其转运至ＣＭＡ受体－溶酶体相关膜蛋白２Ａ(ｌｙｓｏｓｏ￣ｍａｌ￣ａｓｓｏｃｉａｔｅｄｍｅｍｂｒａｎｅｐｒｏｔｅｉｎ２ＡꎬＬＡＭＰ２Ａ)处ꎬ４３３生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.ＬＡＭＰ２Ａ介导ＨＩＦ￣１α亚基转位进入溶酶体腔ꎬ最终被溶酶体中的酸性蛋白酶系降解[１９](图３)ꎮＨＩＦ￣１α亚基的这种稳定性调控机制是非氧依赖的ꎬ其中组成型热休克蛋白７０(ｈｅａｔｓｈｏｃｋｃｏｇｎａｔｅ７０ꎬＨＳＣ７０)和ＬＡＭＰ２Ａ是该机制中的核心组分ꎮ当利用质子泵抑制剂巴伐洛霉素抑制溶酶体膜上的质子泵Ｖ￣ＡＴＰａｓｅ或利用弱碱化合物氯喹中和溶酶体中的酸性环境时ꎬ均可升高ＨＩＦ￣１α亚基的活性和蛋白质水平ꎮ而当过表达促进溶酶体发生的转录因子ＴＦＥＢ或利用强心苷类化合物地高辛激活ＣＭＡ以及血清饥饿或葡萄糖饥饿时ꎬ均导致ＨＩＦ￣１α亚基的活性和蛋白质水平降低[１９] (图３)ꎮ图３㊀由ＣＭＡ介导的溶酶体途径降解ＨＩＦ￣１αＦｉｇ.３㊀ＤｅｇｒａｄａｔｉｏｎｏｆＨＩＦ￣１αｔｈｒｏｕｇｈｌｙｓｏｓｏｍａｌｐａｔｈｗａｙｍｅｄｉａｔｅｄｂｙＣＭＡ.ＣＨＩＰ/ＳＴＵＢ１(ｃａｒｂｏｘｙｔｅｒｍｉｎｕｓｏｆＨｓｐ７０ｉｎ￣ｔｅｒａｃｔｉｎｇｐｒｏｔｅｉｎ)是ＨＩＦ￣１α亚基与ＨＳＣ７０㊁ＬＡＭＰ２Ａ结合所必需的ꎮＳＴＵＢ１既具有分子伴侣结合活性又具有Ｅ３泛素连接酶活性ꎬ其Ｎ端含有３个三十四肽重复(ｔｅｔｒａｔｒｉｃｏｐｅｐｔｉｄｅｒｅｐｅａｔꎬＴＰＲ)结构域ꎬＣ端含有Ｕ￣ｂｏｘ结构域ꎬＴＰＲ结构域通过与胞质中的分子伴侣(ＨＳＰＡ８和ＨＳＰＡ４)相互作用使ＳＴＵＢ１起到辅助分子伴侣活性ꎬＵ￣ｂｏｘ结构域起到的是Ｅ３泛素连接酶活性ꎬ这２种活性均为ＨＩＦ￣１α和ＬＡＭＰ２Ａ结合所必需的[２０]ꎮ另外ꎬ在长期低氧情况下ꎬ热休克蛋白７０(ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ７０ꎬＨＳＰ７０)也可通过招募ＳＴＵＢ１选择性促进ＨＩＦ￣１α亚基被多聚泛素化修饰后由蛋白酶体降解ꎬ这说明ＳＴＵＢ１在ＨＩＦ￣１α亚基的降解机制选择(ＣＭＡ介导的溶酶体降解ꎬ多聚泛素化修饰后蛋白酶体降解)中起到关键作用ꎮ值得注意的是ꎬ虽然ＨＳＰ７０和ＨＳＣ７０在氨基酸序列上具有８６％的相似性[２１]ꎬ但二者与ＨＩＦ￣１α相互作用时的分子机制不同ꎬ事实上ꎬＨＩＦ￣１α的Ｎ端与ＨＳＣ７０的Ｃ端结合ꎬ而ＨＩＦ￣１α的Ｃ端与ＨＳＰ７０的Ｎ端结合[１９]ꎮＨＩＦ￣１α亚基还受到其他非氧依赖机制的调控ꎮ如Ａｄａｍ等[２２]在乳腺癌细胞系ＭＣＦ￣７中发现一种Ｅ３泛素连接酶ＳＩＡＨ１/２(ｓｅｖｅｎ￣ｉｎ￣ａｂｓｅｎｔｉａｈｏｍｏｌｏｇ１/２)以不受Ｏ２水平影响的方式通过降低自身底物ＰＨＤ３的稳定性ꎬ维持ＨＩＦ￣１α亚基的水平ꎬ促进乳腺癌细胞的转移和侵袭ꎮ研究人员在人脑胶质瘤细胞系Ｕ２５１中也观察到ꎬ低氧时ＳＩ￣ＡＨ１使ＰＨＤ３稳定性下降ꎬＨＩＦ￣１α不被降解ꎬ从而促进胶质瘤细胞的转移㊁侵袭[２３]ꎮ此外ꎬ活化的蛋白激酶Ｃ１受体ＲＡＣＫ１㊁精脒/精胺Ｎ１￣乙酰转移酶ＳＳＡＴ１㊁钙调磷酸酶(ｃａｌｃｉｎｅｕｒｉｎ)㊁低氧相关因子(ｈｙｐｏｘｉａ￣ａｓｓｏｃｉａｔｅｄｆａｃｔｏｒꎬＨＡＦ)㊁分化型胚胎软骨发育基因ＳＨＡＲＰ１和ＨＳＰ７０/ＣＨＩＰ(ｃａｒｂｏｘｙｔｅｒｍｉｎｕｓｏｆＨｓｐ７０ｉｎｔｅｒａｃｔｉｎｇｐｒｏｔｅｉｎ)也以非氧依赖的方式调节ＨＩＦ￣１α亚基的蛋白酶体降解[１９]ꎮ３㊀ＨＩＦｓ的转录激活调控ＨＩＦｓ作为转录因子调控人和哺乳动物体内数百种靶基因的表达ꎬ涉及红细胞生成㊁血管生成以及细胞的代谢㊁凋亡㊁迁移和自噬等众多生理过程ꎬ而这些生理过程与肿瘤的发生发展联系紧密ꎮ如自噬在肿瘤发展中具有维持癌变的作用ꎬ而ＨＩＦ￣１α可通过诱导ＢＮＩＰ３表达介导肿瘤细胞的自噬过程[７]ꎻＨＩＦ￣１α还能激活多药耐药基因ＭＤＲ１(ｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅｇｅｎｅ１)的表达ꎬＭＤＲ１编码一种跨膜Ｐ￣糖蛋白(Ｐ￣ｇｌｙｃｏｐｒｏｔｅｉｎꎬＰｇｐ)ꎬ从而将化疗药物泵出肿瘤细胞ꎬ使肿瘤细胞具有化疗抗性ꎻＨＩＦ￣２α可通过减少放疗产生的ＲＯＳ促进肿瘤细胞存活ꎮＨＩＦ￣αｓ亚基转录活性的调控常通过羟基化㊁磷酸化㊁去乙酰化修饰等来实现ꎬ这些修饰作用通过５３３闫东科ꎬ等:低氧诱导因子及其抑制剂研究进展. All Rights Reserved.影响ＨＩＦ￣αｓ亚基对ｐ３００/ＣＢＰ的亲和力㊁与ＨＩＦ￣１β亚基的二聚化㊁与ｐＶＨＬ的相互作用ꎬ对ＨＩＦｓ的转录激活功能起到正向或负向的调控作用ꎮ３.１㊀羟基化ＨＩＦ￣１α和ＨＩＦ￣２α亚基的转录活性受到一种天冬氨酸羟化酶(又称ＨＩＦ￣１α抑制因子ꎬｆａｃｔｏｒ￣ｉｎｈｉｂｉｔｉｎｇＨＩＦ￣１αꎬＦＩＨ￣１)的调控ꎬＦＩＨ￣１的催化功能与ＰＨＤｓ类似ꎬ是Ｏ２㊁α￣酮戊二酸和Ｆｅ２＋依赖的ꎮ常氧时ꎬＦＩＨ￣１可分别羟基化ｈＨＩＦ￣１α亚基Ｃ￣ＴＡＤ结构域中的Ａｓｎ８０３残基和ｈＨＩＦ￣２α亚基Ｃ￣ＴＡＤ结构域中的Ａｓｎ８５１残基[７](图４Ａ)ꎬ阻断ＨＩＦ￣１/２α与ｐ３００/ＣＢＰ的结合ꎬ从而抑制ＨＩＦ￣１和ＨＩＦ￣２的转录激活功能ꎻ而ＨＩＦ￣３α亚基由于缺少Ｃ￣ＴＡＤ结构域ꎬＦＩＨ￣１无法通过羟基化修饰调节其转录活性ꎮ缺氧或有ＣｏＣｌ２㊁ＤＭＯＧ㊁铁离子螯合剂等存在时ꎬＦＩＨ￣１活性被抑制ꎬ未发生羟基化修饰的ＨＩＦ￣１/２α和ＨＩＦ￣１β亚基成功富集ｐ３００/ＣＢＰꎬ从而激活靶基因转录(图４Ｂ)ꎮ图４㊀ＨＩＦ￣１/２的转录激活调控Ｆｉｇ.４㊀ＲｅｇｕｌａｔｉｏｎｏｆｔｒａｎｓｃｒｉｐｔｉｏｎａｌａｃｔｉｖａｔｉｏｎｏｆＨＩＦ￣１/２.３.２㊀磷酸化促分裂原活化蛋白激酶(ｍｉｔｏｇｅｎ￣ａｃｔｉｖａｔｅｄｐｒｏｔｅｉｎｋｉｎａｓｅꎬＭＡＰＫ)途径中的有丝分裂原蛋白激酶ｐ４２/ｐ４４对ＨＩＦ￣１α亚基Ｔｈｒ７９６残基和ＨＩＦ￣２α亚基Ｔｈｒ８４４残基的磷酸化可增强Ｃ￣ＴＡＤ结构域与ＣＢＰ/ｐ３００的相互作用ꎬ使ＨＩＦ￣１和ＨＩＦ￣２的转录活性明显上调ꎮｐ４２/ｐ４４还可通过磷酸化ＨＩＦ￣１α亚基Ｓ６４１和Ｓ６４３残基抑制ＨＩＦ￣１α与出核蛋白ＣＲＭ１的相互作用ꎬ促进ＨＩＦ￣１α在细胞核中的积累[２４]ꎬ从而提高ＨＩＦ￣１的蛋白质水平ꎮ而酪蛋白激酶１(ｃａｓｅｉｎｋｉｎａｓｅ１ꎬＣＫ１)对ＨＩＦ￣１α亚基ＰＡＳ￣Ｂ结构域中的Ｓｅｒ２４７残基的磷酸化作用可抑制ＨＩＦ￣１α与ＨＩＦ￣１β亚基的结合ꎬ减少ＨＩＦ￣１α靶基因的表达[７]ꎮ３.３㊀去乙酰化目前ꎬＮＡＤ＋依赖的组蛋白去乙酰化酶Ｓｉｒｔｕｉｎｓ１(Ｓｉｒｔ１)对ＨＩＦ￣１α亚基活性的调控尚无定论[２５]ꎮ起初ꎬ研究发现Ｓｉｒｔ１特异性对ＨＩＦ￣２α亚基起到去乙酰化作用ꎬ从而增强其转录活性ꎬ而对ＨＩＦ￣１α亚基没有作用ꎮ后来ꎬ有研究表明ꎬ常氧时ꎬＳｉｒｔ１通过去除ＨＩＦ￣１α亚基Ｌｙｓ６７４残基上的乙酰基阻碍其对ｐ３００的富集ꎬ从而抑制ＨＩＦ￣１α亚基的转录活性[２６]ꎻ低氧时ꎬ细胞内氧化还原反应产生的ＮＡＤ＋减少ꎬＳｉｒｔ１去乙酰化酶活性降低ꎬ解除了对ＨＩＦ￣１α的抑制作用ꎬ而ＨＩＦ￣１α亚基Ｌｙｓ６７４残基上的乙酰化修饰由ｐ３００/ＣＢＰ相关因子(ｐ３００/ＣＢＰ￣ａｓｓｏｃｉａｔｅｄｆａｃｔｏｒꎬＰＣＡＦ)负责催化ꎬＰＣＡＦ具有拮抗Ｓｉｒｔ１去乙酰化酶活性的能力[２７]ꎮ再后来研究发现ꎬ肝癌细胞系(ｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒ￣ｃｉｎｏｍａꎬＨＣＣ)中的Ｓｉｒｔ１可促进ＨＩＦ￣１α亚基的积累并对其转录活性起到正向调控作用[２８]ꎮ此外ꎬ低氧环境对Ｓｉｒｔ１活性的影响也不明确ꎮ一种机制认为ꎬ低氧时ꎬ细胞内的ＮＡＤ＋减少ꎬ抑制了Ｓｉｒｔ１的活性ꎻ另一种机制认为ꎬ低氧使Ｓｉｒｔ１的表达以一种低氧诱导因子依赖性的方式上升ꎮ３.４㊀其他调控机制除上述３种修饰作用可调控ＨＩＦ￣αｓ亚基转录活性外ꎬ还有其他机制也可调控ＨＩＦｓ的转录活６３３生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.性ꎮ如哺乳动物Ｓｉｒｔ家族(Ｓｉｒｔ１~７)中的另一成员Ｓｉｒｔ７通过分别与ＨＩＦ￣１α和ＨＩＦ￣２α亚基间的相互作用(这种相互作用被认为是直接的物理作用)ꎬ可在蛋白质水平上对二者起到非氧依赖的负调控作用[２９]ꎮＳｉｒｔ７起到的这种使ＨＩＦ￣１α和ＨＩＦ￣２α降解的调控作用ꎬ具体机制尚不清楚ꎬ但这一过程不需要Ｓｉｒｔ７发挥去乙酰化酶活性ꎬ也不需要Ｏ２/ＰＨＤｓ/ｐＶＨＬ降解途径中的Ｐｒｏ残基被羟基化修饰ꎬ同时也没有蛋白酶体和溶酶体的参与ꎮ抑癌基因ｐ５３对ＨＩＦ￣１α亚基活性也具有调控作用ꎬ主要取决于缺氧的程度和持续时间[３０]ꎮ常氧时ꎬｐ５３和ＨＩＦ￣１α的蛋白质水平较低ꎻ轻度缺氧时ꎬｐ５３处于失活状态ꎬ而ＨＩＦ￣１α开始积累并保持在一定水平ꎬ同时激活抑癌基因ｐ２１的表达ꎬ从而引发短暂的细胞周期停滞和细胞低氧适应ꎻ中度缺氧时ꎬＨＩＦ￣１α亚基的表达水平进一步升高ꎬ使ｐ２１持续表达引发细胞衰老ꎻ严重缺氧时ꎬｐ５３开始逐渐积累并和ＨＩＦ￣１α竞争性地与ｐ３００结合ꎬ从而减弱ＨＩＦ￣１的转录活性ꎬ同时ｐ５３减轻抗凋亡基因(如ｍｉＲ￣１７￣９２)对促凋亡基因(如ＢＩＭ)的抑制作用ꎬ引发细胞凋亡ꎻ极端缺氧时ꎬｐ５３导致ＨＩＦ￣１α经Ｏ２/ＰＨＤｓ/ｐＶＨＬ途径降解ꎬ同时促进细胞凋亡ꎮ此外ꎬＣＳＣ中的ＨＩＦ￣１α和ＨＩＦ￣２α亚基活性还受到磷脂酰肌醇３￣激酶信号途径(ＰＩ３Ｋ￣ＡＫＴ途径)的调节ꎮ该途径通过对ＨＩＦ￣１α亚基的正向调控激活ＣＳＣ存活相关基因(如糖酵解酶类基因)ꎬ同时抑制抑癌基因ｐ５３的活性ꎬ从而促进ＣＳＣ的存活ꎻ该途径还可通过激活ＨＩＦ￣２α亚基提高其下游ＣＳＣ干性相关基因Ｏｃｔ￣４㊁Ｓｏｘ￣２等的表达水平ꎬ促进ＣＳＣ的干性维持[３１]ꎮ４㊀ＨＩＦｓ抑制剂目前ꎬ已将ＨＩＦｓ抑制剂作为靶向抗癌药物的重要研究内容ꎬ大多数ＨＩＦｓ抑制剂的靶向目标为ＨＩＦ￣１α和ＨＩＦ￣２αꎬ尚未开发出针对ＨＩＦ￣３α的特异性抑制剂[６]ꎮ４.１㊀影响ＨＩＦ￣αｍＲＮＡ或ＨＩＦ￣α蛋白合成的抑制剂人工合成的反义寡脱氧核苷酸ＥＺＮ￣２９６８含有１６个与ｈＨＩＦ￣１αｍＲＮＡ１００％互补的核苷酸残基ꎬ其可以剂量依赖性方式下调ｈＨＩＦ￣１α亚基的表达ꎬ且在浓度为５ｎｍｏｌ/Ｌ时具有完全抑制活性ꎮＥＺＮ￣２９６８与ＨＩＦ￣２αｍＲＮＡ具有３个碱基对错配ꎬ故对ＨＩＦ￣２α亚基的抑制作用较弱ꎮ针对ＥＺＮ￣２９６８的Ⅰ期临床试验肿瘤活检结果表明ꎬＥＺＮ￣２９６８降低了ＨＩＦ￣１α亚基和靶基因的ｍＲＮＡ水平[３２]ꎮｍｉｃｒｏＲＮＡｓ(ｍｉＲＮＡｓ)可通过与ＨＩＦ￣αｍＲＮＡ的相互作用来调控ＨＩＦ￣α的合成ꎮ如ｍｉＲ￣１４５[３３]和ｍｉＲ￣５５８[３４]通过碱基互补配对原则分别与ＨＩＦ￣２αｍＲＮＡ的３ᶄ端非编码区和５ᶄ端非编码区结合来抑制其转录翻译过程ꎮＨｕｔｔ等[３５]在ＨＣＣ细胞中发现组蛋白去乙酰化酶抑制剂(ｈｉｓｔｏｎｅｄｅａｃｅｔｙｌａｓｅｓｉｎｈｉｂｉｔｏｒｓꎬＨＤＡＣｉｓ)伏立诺他能通过抑制ＨＤＡＣ９以一种ｅＩＦ３Ｇ(真核翻译起始因子ꎬｅｕｋａｒｙｏｔｉｃｔｒａｎｓｌａｔｉｏｎｉｎｉｔｉａｔｉｏｎｆａｃｔｏｒ)依赖的翻译机制降低ＨＩＦ￣１α亚基的蛋白质水平ꎮ伏立诺他和另一种ＨＤＡＣｉｓ罗米地辛已被美国ＦＤＡ批准用于治疗皮肤Ｔ细胞淋巴癌ꎮ喜树碱的半合成类似物拓扑替康(ｔｏｐｏｔｅｃａｎꎬＴＰＴ)是拓扑异构酶Ⅰ(ｔｏｐｏｉｓｏｍｅｒａｓｅＩꎬＴｏｐＩ)的抑制剂ꎬ可在翻译水平上抑制ＨＩＦ￣１α亚基的产生ꎬ喜树碱类药物可用于小细胞肺癌和卵巢癌等的临床治疗[３６]ꎮ雌激素代谢物２￣甲氧基雌二醇(２￣Ｍｅｔｈｏｘｙｅｓｔｒａｄｉｏｌꎬ２ＭＥ２)可通过抑制ＨＩＦ￣１α和ＨＩＦ￣２α亚基的翻译合成过程以及二者的核易位过程来阻碍肿瘤生长和血管生成[３７]ꎮ此外ꎬＳｈｕｋｌａ等[３８]发现ＨＩＦ￣１α通过上调胞苷三磷酸合成(ｃｙｔｉｄｉｎｅｔｒｉｐｈｏｓｐｈａｔｅｓｙｎｔｈａｓｅꎬＣＴＰＳ１)和转酮醇酶(ｔｒａｎｓｋｅｔｏｌａｓｅꎬＴＫＴ)的表达介导胰腺癌细胞对吉西他滨的耐药性ꎬ而当利用地高辛抑制ＨＩＦ￣１α亚基的翻译过程后ꎬ胰腺癌细胞对吉西他滨的敏感性增强ꎮ４.２㊀影响ＨＩＦ￣α亚基稳定性或二聚化的抑制剂格尔德霉素(ｇｅｌｄａｎａｍｙｃｉｎꎬＧＤＭ)及其合成衍生物１７￣烯丙基氨基格尔德霉素(１７￣ａｌｌｙｌａｍｉｎｏ￣１７￣ｄｅｍｅｔｈｏｘｙｇｅｌｄａｎａｍｙｃｉｎꎬ１７￣ＡＡＧ)可通过抑制热休克蛋白９０(ｈｅａｔ￣ｓｈｏｃｋｐｒｏｔｅｉｎ９０ꎬＨＳＰ９０)的活性使ＨＩＦ￣α亚基因无法正确折叠和定位ꎬ从而以不依赖ｐＶＨＬ的方式降解ꎮ另一种小分子ＨＳＰ９０抑制剂ＥＣ１５４相较于１７￣ＡＡＧ具有更强的７３３闫东科ꎬ等:低氧诱导因子及其抑制剂研究进展. All Rights Reserved.抑制ＨＳＰ９０活性的能力[３９]ꎮＨＩＦ￣αｓ和ＨＩＦ￣１β亚基中的ＰＡＳ结构域参与了ＨＩＦｓ异源二聚体的组装ꎮ因此ꎬ靶向ＰＡＳ结构域的小分子可影响ＨＩＦ￣αｓ与ＨＩＦ￣１β的二聚化ꎮ消毒灭菌剂吖啶黄素(ａｃｒｉｆｌａｖｉｎｅ)通过结合至ＨＩＦ￣αｓ亚基ＰＡＳ￣Ｂ结构域和ＨＩＦ￣１β亚基ＰＡＳ￣Ａ结构域相结合的界面处ꎬ破坏ＨＩＦｓ异源二聚体的稳定性[４０]ꎮ环肽抑制剂(环￣ＣＬＬＦＶＹ)选择性作用于ＨＩＦ￣１α亚基ＰＡＳ￣Ｂ结构域ꎬ从而破坏ＨＩＦ￣１的二聚化过程ꎬ而对ＨＩＦ￣２的二聚化过程没有影响[４１]ꎻ化合物ＰＴ２３８５选择性作用于ＨＩＦ￣２α亚基ＰＡＳ￣Ｂ结构域ꎬ而对ＨＩＦ￣１没有影响[４２]ꎻ双环化合物ＯＸ３可结合至ＨＩＦ￣２α亚基ＰＡＳ￣Ｂ结构域的疏水口袋内ꎬ影响ＨＩＦ￣２的构象稳定和ＨＲＥ序列结合活性ꎬ但对ＨＩＦ￣１几乎没有影响[５]ꎮ４.３㊀影响ＨＩＦｓ与ＤＮＡ结合的抑制剂ＨＩＦｓ主要是通过与靶基因中的ＨＲＥ序列结合发挥转录激活作用ꎮ利用染色质免疫共沉淀技术(ＣｈＩＰａｓｓａｙ)对人脑胶质瘤细胞系Ｕ２５１进行体外研究证实ꎬ棘霉素(ｅｃｈｉｎｏｍｙｃｉｎ)可特异性抑制ＨＩＦ￣１与ＶＥＧＦ启动子区的ＨＲＥ序列(５ᶄ￣ＴＡＣＧＴＧ￣３ᶄ)结合ꎬ从而抑制缺氧诱导的ＶＥＧＦ的表达[４３]ꎬ但棘霉素的临床试验效果欠佳ꎮ此外ꎬ靶向ＨＲＥ序列的ＨＩＦ￣１抑制剂还有聚酰胺类化合物㊁多柔比星和柔红霉素[４４]ꎮ４.４㊀影响ＨＩＦｓ转录复合物形成的抑制剂来自真菌黑毛菌属毛壳菌的毛壳菌素(ｃｈｅｔ￣ｏｍｉｎ)可通过作用于ｐ３００ＣＨ１结构域中的锌结合位点使Ｚｎ２＋外排ꎬ改变ＣＨ１结构域的构象ꎬ从而破坏ｐ３００与ＨＩＦ￣１α的相互作用[４５]ꎮＲｅｅｃｅ等[４６]证实毛壳菌素以剂量依赖性方式使分泌型ＶＥＧＦ㊁乳酸脱氢酶Ａ(ｌａｃｔａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅＡꎬＬＤＨＡ)和烯醇酶１(ｅｎｏｌａｓｅ１ꎬＥＮＯ１)的表达下降ꎬ最终导致鼠前列腺癌异种移植细胞的生长受到明显抑制ꎮ硼替佐米的抗肿瘤活性是通过增强天冬氨酸羟化酶ＦＩＨ与ＨＩＦ￣１α的结合ꎬ破坏ＨＩＦ￣１α对ｐ３００的富集作用[４７]ꎮ此外ꎬ抗血小板凝集剂ＹＣ￣１和噻唑烷酮化合物也都是通过破坏ＨＩＦ￣αｓ与ｐ３００间的相互作用ꎬ来抑制ＨＩＦｓ对靶基因的转录激活活性ꎬ而根据ＹＣ￣１的结构设计合成出的化合物ＣＪ￣３ｋ也可高效抑制ＨＩＦ￣１α的活性[４８]ꎮ５㊀展望目前ꎬ大多数靶向特异性生长因子及其受体或通路的药物在应用于癌症治疗的临床试验后ꎬ最终会使癌症产生相应抗性ꎬ而实体肿瘤内部的异质性和克隆进化也可能使肿瘤获得相应抗性ꎮ相比较而言ꎬ因为ＨＩＦｓ参与了肿瘤细胞分化㊁肿瘤细胞代谢重编程㊁肿瘤血管生成并促进了肿瘤转移和治疗抗性ꎬ所以靶向肿瘤组织的缺氧表型被认为是治疗癌症的更为有效的方法ꎮ如前所述ꎬＨＩＦ￣１α㊁ＨＩＦ￣２α和ＨＩＦ￣３α在蛋白质结构㊁稳定性调控和转录激活调控上均有相似之处ꎬ但三者在不同类型肿瘤的发生㊁发展中表现出功能关系上的复杂性ꎮ如Ｊｉａｎｇ等[４９]发现ＨＩＦ￣１α和ＨＩＦ￣２α在宫颈癌细胞系ＣａＳｋｉ的存活㊁凋亡和细胞周期中具有类似的作用ꎬ在单一抑制ＨＩＦ￣１α或ＨＩＦ￣２α的表达时ꎬ均可将ＣａＳｋｉ的细胞周期阻断在Ｇ１期ꎮ再如ꎬ对膀胱癌Ｔ２４细胞系的体外研究表明ꎬ长时间低氧情况下ꎬＨＡＦ表达水平升高并起到Ｅ３泛素连接酶的作用ꎬ同时激活ＮＦ￣κＢ途径ꎬ使ＨＩＦ￣１α以非氧依赖的方式经多聚泛素化蛋白酶体途径降解ꎻ而此时ＨＩＦ￣２α的表达补偿性增加ꎬ从而使Ｔ２４细胞加速恶化并有利于Ｔ２４干细胞标志物的维持[５０]ꎮ这表明ＨＩＦ￣１α和ＨＩＦ￣２α之间存在代偿性机制ꎮ另外ꎬ过表达ＨＩＦ￣１α亚基可减慢ｐＶＨＬ缺陷型肾细胞癌(ｒｅｎａｌｃｅｌｌｃａｒｃｉｎｏｍａꎬＲＣＣ)异种移植细胞的生长ꎬ而过表达ＨＩＦ￣２α亚基可促进ＲＣＣ移植细胞的生长ꎮ这表明在一些肿瘤中ꎬＨＩＦ￣１α和ＨＩＦ￣２α起相反作用[５１]ꎮ因此ꎬ根据不同肿瘤研发出针对不同ＨＩＦｓ家族成员的特异性靶向抑制剂药物应是今后的一个主攻方向ꎮ同时ꎬ为了提高临床治疗效果ꎬＨＩＦｓ抑制剂应与放疗㊁化疗和免疫治疗等联合应用于临床试验ꎮ参㊀考㊀文㊀献[１]㊀ＰｉｎｈｅｉｒｏＣꎬＭｉｒａｎｄａ￣ＧｏｎｃａｌｖｅｓＶꎬＬｏｎｇａｔｔｏ￣ＦｉｌｈｏＡꎬｅｔａｌ..Ｔｈｅｍｅｔａｂｏｌｉｃｍｉｃｒｏｅｎｖｉｒｏｎｍｅｎｔｏｆｍｅｌａｎｏｍａｓ:ＰｒｏｇｎｏｓｔｉｃｖａｌｕｅｏｆＭＣＴ１ａｎｄＭＣＴ４[Ｊ].ＣｅｌｌＣｙｃｌｅꎬ２０１６ꎬ１５(１１):１４６２－１４７０.[２]㊀ＳｅｒｏｃｋｉＭꎬＢａｒｔｏｓｚｅｗｓｋａＳꎬＪａｎａｓｚａｋ￣ＪａｓｉｅｃｋａＡꎬｅｔａｌ..ｍｉＲ￣８３３生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.ＮＡｓｒｅｇｕｌａｔｅｔｈｅＨＩＦｓｗｉｔｃｈｄｕｒｉｎｇｈｙｐｏｘｉａ:Ａｎｏｖｅｌｔｈｅｒａｐｅｕｔｉｃｔａｒｇｅｔ[Ｊ].Ａｎｇｉｏｇｅｎｅｓｉｓꎬ２０１８ꎬ２１(２):１８３－２０２. [３]㊀ＫｌａｕｓＡꎬＦａｔｈｉＯꎬＴａｔｊａｎａＴＷꎬｅｔａｌ..Ｅｘｐｒｅｓｓｉｏｎｏｆｈｙｐｏｘｉａ￣ａｓｓｏｃｉａｔｅｄｐｒｏｔｅｉｎＨＩＦ￣１αｉｎｆｏｌｌｉｃｕｌａｒｔｈｙｒｏｉｄｃａｎｃｅｒｉｓａｓｓｏｃｉ￣ａｔｅｄｗｉｔｈｄｉｓｔａｎｔｍｅｔａｓｔａｓｉｓ[Ｊ].Ｐａｔｈｏｌ.Ｏｎｃｏｌ.Ｒｅｓ.ꎬ２０１８ꎬ２４(２):２８９－２９６.[４]㊀ＺｈｕＧＨꎬＨｕａｎｇＣꎬＦｅｎｇＺＺꎬｅｔａｌ..Ｈｙｐｏｘｉａ￣ｉｎｄｕｃｅｄｓｎａｉｌｅｘｐｒｅｓｓｉｏｎｔｈｒｏｕｇｈｔｒａｎｓｃｒｉｐｔｉｏｎａｌｒｅｇｕｌａｔｉｏｎｂｙＨＩＦ￣１ａｌｐｈａｉｎｐａｎｃｒｅａｔｉｃｃａｎｃｅｒｃｅｌｌｓ[Ｊ].Ｄｉｇｅｓｔ.Ｄｉｓ.Ｓｃｉ.ꎬ２０１３ꎬ５８(１２):３５０３－３５１５.[５]㊀ＷｉｌｋｉｎｓＳＥꎬＡｂｂｏｕｄＭＩꎬＨａｎｃｏｃｋＲＬꎬｅｔａｌ..Ｔａｒｇｅｔｉｎｇｐｒｏｔｅｉｎ￣ｐｒｏｔｅｉｎｉｎｔｅｒａｃｔｉｏｎｓｉｎｔｈｅＨＩＦｓｙｓｔｅｍ[Ｊ].ＣｈｅｍＭｅｄ￣Ｃｈｅｍꎬ２０１６ꎬ１１(８):７７３－７８６.[６]㊀ＤｕａｎＣ.Ｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ３ｂｉｏｌｏｇｙ:Ｃｏｍｐｌｅｘｉｔｉｅｓａｎｄｅｍｅｒｇｉｎｇｔｈｅｍｅｓ[Ｊ].Ａｍ.Ｊ.Ｐｈｙｓｉｏｌ.ＣｅｌｌＰｈ.ꎬ２０１６ꎬ３１０(４):Ｃ２６０－Ｃ２６９.[７]㊀Ｓａｉｎｔ￣ＭａｒｔｉｎＡꎬＣａｓｔａñｅｄａ￣ＰａｔｌáｎＭꎬＲｏｂｌｅｓ￣ＦｌｏｒｅｓＭ.Ｔｈｅｒｏｌｅｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓｉｎｃａｎｃｅｒｒｅｓｉｓｔａｎｃｅ[Ｊ].Ｊ.ＣｅｌｌＳｉｇｎａｌ.ꎬ２０１７ꎬ２:１５４.[８]㊀ＫａｌｌｉｏＰＪ.Ｓｉｇｎａｌｔｒａｎｓｄｕｃｔｉｏｎｉｎｈｙｐｏｘｉｃｃｅｌｌｓ:Ｉｎｄｕｃｉｂｌｅｎｕ￣ｃｌｅａｒｔｒａｎｓｌｏｃａｔｉｏｎａｎｄｒｅｃｒｕｉｔｍｅｎｔｏｆｔｈｅＣＢＰ/ｐ３００ｃｏａｃｔｉｖａｔｏｒｂｙｔｈｅｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ａｌｐｈａ[Ｊ].ＥＭＢＯＪ.ꎬ２０１４ꎬ１７(２２):６５７３－６５８６.[９]㊀ＹａｏＱꎬＺｈａｎｇＰꎬＬｕＬꎬｅｔａｌ..ＮｕｃｌｅａｒｌｏｃａｌｉｚａｔｉｏｎｏｆＨｉｆ￣３ａｌｐｈａｒｅｑｕｉｒｅｓｔｗｏｒｅｄｕｎｄａｎｔＮＬＳｍｏｔｉｆｓｉｎｉｔｓｕｎｉｑｕｅＣ￣ｔｅｒ￣ｍｉｎａｌｒｅｇｉｏｎ[Ｊ].ＦＥＢＳＬｅｔｔ.ꎬ２０１８ꎬ５９２(１６):２７６９－２７７５. [１０]㊀ＸｉａｎｇＸꎬＫｙｌｉｅＪꎬＧｕｎｓｅｌｉＯꎬｅｔａｌ..ＨＩＦ￣３α１ｐｒｏｍｏｔｅｓｃｏｌｏｒ￣ｅｃｔａｌｔｕｍｏｒｃｅｌｌｇｒｏｗｔｈｂｙａｃｔｉｖａｔｉｏｎｏｆＪＡＫ￣ＳＴＡＴ３ｓｉｇｎａｌｉｎｇ[Ｊ].Ｏｎｃｏｔａｒｇｅｔꎬ２０１６ꎬ７(１０):１１５６７－１１５７９.[１１]㊀ＰｅｎｇＺꎬＹａｎＢꎬＬｉｎｇＬꎬｅｔａｌ..Ａｎｏｘｙｇｅｎ￣ｉｎｓｅｎｓｉｔｉｖｅＨｉｆ￣３αｉｓｏｆｏｒｍｉｎｈｉｂｉｔｓＷｎｔｓｉｇｎａｌｉｎｇｂｙｄｅｓｔａｂｉｌｉｚｉｎｇｔｈｅｎｕｃｌｅａｒｂ￣ｃａｔｅｎｉｎｃｏｍｐｌｅｘ[Ｊ].ｅＬｉｆｅꎬ２０１６ꎬ１４(５):ｅ０８９９６. [１２]㊀ＹａｎｇＳＬꎬＷｕＣꎬＸｉｏｎｇＺＦꎬｅｔａｌ..Ｐｒｏｇｒｅｓｓｏｎｈｙｐｏｘｉａ￣ｉｎ￣ｄｕｃｉｂｌｅｆａｃｔｏｒ￣３:Ｉｔｓｓｔｒｕｃｔｕｒｅꎬｇｅｎｅｒｅｇｕｌａｔｉｏｎａｎｄｂｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎ[Ｊ].Ｍｏｌ.Ｍｅｄ.Ｒｅｐ.ꎬ２０１５ꎬ１２(２):２４１１－２４１６. [１３]㊀ＷｉｇｅｒｕｐＣꎬＰåｈｌｍａｎＳꎬＢｅｘｅｌｌＤ.Ｔｈｅｒａｐｅｕｔｉｃｔａｒｇｅｔｉｎｇｏｆｈｙ￣ｐｏｘｉａａｎｄｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓｉｎｃａｎｃｅｒ[Ｊ].Ｐｈａｒｍａｃｏｌ.Ｔｈｅｒａｐｅｕｔ.ꎬ２０１６ꎬ１６４(１):１５２－１６９.[１４]㊀ＨｕＹＹꎬＦｕＬＡꎬＬｉＳＺꎬｅｔａｌ..Ｈｉｆ￣１αａｎｄＨｉｆ￣２αｄｉｆｆｅｒｅｎｔｉ￣ａｌｌｙｒｅｇｕｌａｔｅＮｏｔｃｈｓｉｇｎａｌｉｎｇｔｈｒｏｕｇｈｃｏｍｐｅｔｉｔｉｖｅｉｎｔｅｒａｃｔｉｏｎｗｉｔｈｔｈｅｉｎｔｒａｃｅｌｌｕｌａｒｄｏｍａｉｎｏｆＮｏｔｃｈｒｅｃｅｐｔｏｒｓｉｎｇｌｉｏｍａｓｔｅｍｃｅｌｌｓ[Ｊ].ＣａｎｃｅｒＬｅｔｔ.ꎬ２０１４ꎬ３４９(１):６７－７６.[１５]㊀ＺｈａｏＪꎬＤｕＦꎬＳｈｅｎＧꎬｅｔａｌ..Ｔｈｅｒｏｌｅｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣２ｉｎｄｉｇｅｓｔｉｖｅｓｙｓｔｅｍｃａｎｃｅｒｓ[Ｊ].ＣｅｌｌＤｅａｔｈＤｉｓ.ꎬ２０１５ꎬ６:ｅ１６００.[１６]㊀ＷｉｅｌｏｃｋｘＢꎬＧｒｉｎｅｎｋｏＴꎬＭｉｒｔｓｃｈｉｎｋＰꎬｅｔａｌ..Ｈｙｐｏｘｉａｐａｔｈｗａｙｐｒｏｔｅｉｎｓｉｎｎｏｒｍａｌａｎｄｍａｌｉｇｎａｎｔｈｅｍａｔｏｐｏｉｅｓｉｓ[Ｊ].Ｃｅｌｌｓꎬ２０１９ꎬ８(２):１５５－１６５.[１７]㊀ＳｅｍｅｎｚａＧＬ.Ｔａｒｇｅｔｉｎｇｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ１ｔｏｓｔｉｍｕｌａｔｅｔｉｓｓｕｅｖａｓｃｕｌａｒｉｚａｔｉｏｎ[Ｊ].Ｊ.Ｉｎｖｅｓｔ.Ｍｅｄ.ꎬ２０１６ꎬ６４(２):３６１－３６３.[１８]㊀ＸｉｌｏｕｒｉＭꎬＳｔｅｆａｎｉｓＬ.Ｃｈａｐｅｒｏｎｅｍｅｄｉａｔｅｄａｕｔｏｐｈａｇｙｉｎａｇｉｎｇ:Ｓｔａｒｖｅｔｏｐｒｏｓｐｅｒ[Ｊ].ＡｇｅｉｎｇＲｅｓ.Ｒｅｖ.ꎬ２０１６ꎬ３２:１３－２１. [１９]㊀ＨｕｂｂｉＭＥꎬＨｕＨꎬＫｓｈｉｔｉｚꎬｅｔａｌ..Ｃｈａｐｅｒｏｎｅ￣ｍｅｄｉａｔｅｄａｕｔｏ￣ｐｈａｇｙｔａｒｇｅｔｓｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ａｌｐｈａ(ＨＩＦ￣１ａｌｐｈａ)ｆｏｒｌｙｓｏｓｏｍａｌｄｅｇｒａｄａｔｉｏｎ[Ｊ].Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.ꎬ２０１３ꎬ２８８(１５):１０７０３－１０７１４.[２０]㊀ＦｅｒｒｅｉｒａＪＶꎬＦｏｆｏＨꎬＢｅｊａｒａｎｏＥꎬｅｔａｌ..ＳＴＵＢ１/ＣＨＩＰｉｓｒｅ￣ｑｕｉｒｅｄｆｏｒＨＩＦ１Ａｄｅｇｒａｄａｔｉｏｎｂｙｃｈａｐｅｒｏｎｅ￣ｍｅｄｉａｔｅｄａｕｔｏｐｈａｇｙ[Ｊ].Ａｕｔｏｐｈａｇｙꎬ２０１３ꎬ９(９):１３４９－１３６６.[２１]㊀ＳｏｓｓＳＥꎬＲｏｓｅＫＬꎬＨｉｌｌＳꎬｅｔａｌ..ＢｉｏｃｈｅｍｉｃａｌａｎｄｐｒｏｔｅｏｍｉｃａｎａｌｙｓｉｓｏｆｕｂｉｑｕｉｔｉｎａｔｉｏｎｏｆＨｓｃ７０ａｎｄＨｓｐ７０ｂｙｔｈｅＥ３ｌｉｇａｓｅＣＨＩＰ[Ｊ].ＰＬｏＳＯＮＥꎬ２０１５ꎬ１０(５):ｅ０１２８２４０.[２２]㊀ＡｄａｍＭＧꎬＭａｔｔＳꎬＣｈｒｉｓｔｉａｎＳꎬｅｔａｌ..ＳＩＡＨｕｂｉｑｕｉｔｉｎｌｉｇａｓｅｓｒｅｇｕｌａｔｅｂｒｅａｓｔｃａｎｃｅｒｃｅｌｌｍｉｇｒａｔｉｏｎａｎｄｉｎｖａｓｉｏｎｉｎｄｅｐｅｎｄｅｎｔｏｆｔｈｅｏｘｙｇｅｎｓｔａｔｕｓ[Ｊ].ＣｅｌｌＣｙｃｌｅꎬ２０１５ꎬ１４(２３):３７３４－３７４７.[２３]㊀ＳｈｉＨꎬＺｈｅｎｇＢꎬＷｕＹꎬｅｔａｌ..ＵｂｉｑｕｉｔｉｎｌｉｇａｓｅＳｉａｈ１ｐｒｏｍｏｔｅｓｔｈｅｍｉｇｒａｔｉｏｎａｎｄｉｎｖａｓｉｏｎｏｆｈｕｍａｎｇｌｉｏｍａｃｅｌｌｓｂｙｒｅｇｕｌａｔｉｎｇＨＩＦ￣１αｓｉｇｎａｌｉｎｇｕｎｄｅｒｈｙｐｏｘｉａ[Ｊ].Ｏｎｃｏｌ.Ｒｅｐ.ꎬ２０１５ꎬ３３(３):１１８５－１１９０.[２４]㊀ＫｉｅｔｚｍａｎｎＴꎬＭｅｎｎｅｒｉｃｈＤꎬＤｉｍｏｖａＥＹ.Ｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓ(ＨＩＦｓ)ａｎｄｐｈｏｓｐｈｏｒｙｌａｔｉｏｎ:Ｉｍｐａｃｔｏｎｓｔａｂｉｌｉｔｙꎬｌｏｃａｌ￣ｉｚａｔｉｏｎꎬａｎｄｔｒａｎｓａｃｔｉｖｉｔｙ[Ｊ].Ｆｒｏｎｔ.ＣｅｌｌＤｅｖ.Ｂｉｏｌ.ꎬ２０１６ꎬ４:１１.[２５]㊀ＪｏｏＨＹꎬＹｕｎＭꎬＪｅｏｎｇＪꎬｅｔａｌ..ＳＩＲＴ１ｄｅａｃｅｔｙｌａｔｅｓａｎｄｓｔａ￣ｂｉｌｉｚｅｓｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ａｌｐｈａ(ＨＩＦ￣１ａｌｐｈａ)ｖｉａｄｉｒｅｃｔｉｎｔｅｒａｃｔｉｏｎｓｄｕｒｉｎｇｈｙｐｏｘｉａ[Ｊ].Ｂｉｏｃｈｅｍ.Ｂｉｏｐｈ.Ｒｅｓ.Ｃｏ.ꎬ２０１５ꎬ４６２(４):２９４－３００.[２６]㊀ＬｅｅＳＤꎬＫｉｍＷꎬＪｅｏｎｇＪＷꎬｅｔａｌ..ＡＫ￣１ꎬａＳＩＲＴ２ｉｎｈｉｂｉｔｏｒꎬｄｅｓｔａｂｉｌｉｚｅｓＨＩＦ￣１ａｌｐｈａａｎｄｄｉｍｉｎｉｓｈｅｓｉｔｓｔｒａｎｓｃｒｉｐ￣ｔｉｏｎａｌａｃｔｉｖｉｔｙｄｕｒｉｎｇｈｙｐｏｘｉａ[Ｊ].ＣａｎｃｅｒＬｅｔｔ.ꎬ２０１６ꎬ３７３(１):１３８－１４５.[２７]㊀ＳｕｂｂａｒａｍａｉａｈＫꎬＩｙｅｎｇａｒＮＭꎬＭｏｒｒｏｗＭꎬｅｔａｌ..ＰｒｏｓｔａｇｌａｎｄｉｎＥ２ｄｏｗｎ￣ｒｅｇｕｌａｔｅｓｓｉｒｔｕｉｎ１(ＳＩＲＴ１)ꎬｌｅａｄｉｎｇｔｏｅｌｅｖａｔｅｄｌｅｖｅｌｓｏｆａｒｏｍａｔａｓｅꎬｐｒｏｖｉｄｉｎｇｉｎｓｉｇｈｔｓｉｎｔｏｔｈｅｏｂｅｓｉｔｙ￣ｂｒｅａｓｔｃａｎｃｅｒｃｏｎｎｅｃｔｉｏｎ[Ｊ].Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.ꎬ２０１９ꎬ２９４(１):３６１－３７１.[２８]㊀ＣａｌｇａｎｉＡꎬＤｅｌｌｅＭｏｎａｃｈｅＳꎬＣｅｓａｒｅＰꎬｅｔａｌ..Ｌｅｐｔｉｎｃｏｎｔｒｉｂ￣ｕｔｅｓｔｏｌｏｎｇ￣ｔｅｒｍｓｔａｂｉｌｉｚａｔｉｏｎｏｆＨＩＦ￣１ａｌｐｈａｉｎｃａｎｃｅｒｃｅｌｌｓｓｕｂｊｅｃｔｅｄｔｏｏｘｙｇｅｎｌｉｍｉｔｉｎｇｃｏｎｄｉｔｉｏｎｓ[Ｊ].ＣａｎｃｅｒＬｅｔｔ.ꎬ２０１６ꎬ３７６(１):１－９.[２９]㊀ＨｕｂｂｉＭＥꎬＨｕＨꎬＫｓｈｉｔｉｚꎬｅｔａｌ..Ｓｉｒｔｕｉｎ￣７ｉｎｈｉｂｉｔｓｔｈｅａｃｔｉｖ￣ｉｔｙｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓ[Ｊ].Ｊ.Ｂｉｏｌ.Ｃｈｅｍ.ꎬ２０１３ꎬ２８８(２９):２０７６８－２０７７５.[３０]㊀ＺｈｏｕＣＨꎬＺｈａｎｇＸＰꎬＬｉｕＦꎬｅｔａｌ..ＭｏｄｅｌｉｎｇｔｈｅｉｎｔｅｒｐｌａｙｂｅｔｗｅｅｎｔｈｅＨＩＦ￣１ａｎｄｐ５３ｐａｔｈｗａｙｓｉｎｈｙｐｏｘｉａ[Ｊ].Ｓｃｉ.Ｒｅｐ.ꎬ２０１５ꎬ５(１):１３８３４－１３８４３.[３１]㊀ＳｃｈｏｎｉｎｇＪＰꎬＭｏｎｔｅｉｒｏＭꎬＧｕＷ.Ｄｒｕｇｒｅｓｉｓｔａｎｃｅａｎｄｃａｎｃｅｒｓｔｅｍｃｅｌｌｓ:Ｔｈｅｓｈａｒｅｄｂｕｔｄｉｓｔｉｎｃｔｒｏｌｅｓｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓＨＩＦ１ａｌｐｈａａｎｄＨＩＦ２ａｌｐｈａ[Ｊ].Ｃｌｉｎ.Ｅｘｐ.Ｐｈａｒｍａｃｏｌ.Ｐｈｙｓｉｏｌ.ꎬ２０１７ꎬ４４(２):１５３－１６１.[３２]㊀ＪｅｏｎｇＷꎬＲａｐｉｓａｒｄａＡꎬＰａｒｋＳＲꎬｅｔａｌ..ＰｉｌｏｔｔｒｉａｌｏｆＥＺＮ￣２９６８ꎬａｎａｎｔｉｓｅｎｓｅｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｉｎｈｉｂｉｔｏｒｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ａｌｐｈａ(ＨＩＦ￣１ａｌｐｈａ)ꎬｉｎｐａｔｉｅｎｔｓｗｉｔｈｒｅｆｒａｃ￣ｔｏｒｙｓｏｌｉｄｔｕｍｏｒｓ[Ｊ].ＣａｎｃｅｒＣｈｅｍｏｔｈ.Ｐｈａｒｍ.ꎬ２０１４ꎬ７３(２):３４３－３４８.[３３]㊀ＺｈａｎｇＨꎬＰｕＪꎬＱｉＴꎬｅｔａｌ..ＭｉｃｒｏＲＮＡ￣１４５ｉｎｈｉｂｉｔｓｔｈｅｇｒｏｗｔｈꎬｉｎｖａｓｉｏｎꎬｍｅｔａｓｔａｓｉｓａｎｄａｎｇｉｏｇｅｎｅｓｉｓｏｆｎｅｕｒｏｂｌａｓｔｏｍａｃｅｌｌｓｔｈｒｏｕｇｈｔａｒｇｅｔｉｎｇｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ２ａｌｐｈａ[Ｊ].９３３闫东科ꎬ等:低氧诱导因子及其抑制剂研究进展. All Rights Reserved.Ｏｎｃｏｇｅｎｅꎬ２０１４ꎬ３３(３):３８７－３９７.[３４]㊀ＱｕＨꎬＺｈｅｎｇＬꎬＳｏｎｇＨꎬｅｔａｌ..ｍｉｃｒｏＲＮＡ￣５５８ｆａｃｉｌｉｔａｔｅｓｔｈｅｅｘｐｒｅｓｓｉｏｎｏｆｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ２ａｌｐｈａｔｈｒｏｕｇｈｂｉｎｄｉｎｇｔｏ５ᶄ￣ｕｎｔｒａｎｓｌａｔｅｄｒｅｇｉｏｎｉｎｎｅｕｒｏｂｌａｓｔｏｍａ[Ｊ].Ｏｎｃｏｔａｒｇｅｔꎬ２０１６ꎬ７(２６):４０６５７－４０６７３.[３５]㊀ＨｕｔｔＤＭꎬＲｏｔｈＤＭꎬＶｉｇｎａｕｄＨꎬｅｔａｌ..Ｔｈｅｈｉｓｔｏｎｅｄｅａｃｅｔｙ￣ｌａｓｅｉｎｈｉｂｉｔｏｒꎬｖｏｒｉｎｏｓｔａｔꎬｒｅｐｒｅｓｓｅｓｈｙｐｏｘｉａｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ１ａｌｐｈａｅｘｐｒｅｓｓｉｏｎｔｈｒｏｕｇｈｔｒａｎｓｌａｔｉｏｎａｌｉｎｈｉｂｉｔｉｏｎ[Ｊ].ＰＬｏＳＯＮＥꎬ２０１４ꎬ９(８):ｅ１０６２２４.[３６]㊀ＣｏｌｔｅｌｌａＮꎬＶａｌｓｅｃｃｈｉＲꎬＰｏｎｅｎｔｅＭꎬｅｔａｌ..Ｓｙｎｅｒｇｉｓｔｉｃｌｅｕｋｅ￣ｍｉａｅｒａｄｉｃａｔｉｏｎｂｙｃｏｍｂｉｎｅｄｔｒｅａｔｍｅｎｔｗｉｔｈｒｅｔｉｎｏｉｃａｃｉｄａｎｄＨＩＦｉｎｈｉｂｉｔｉｏｎｂｙＥＺＮ￣２２０８(ＰＥＧ￣ＳＮ３８)ｉｎｐｒｅｃｌｉｎｉｃａｌｍｏｄｅｌｓｏｆＰＭＬ￣ＲＡＲαａｎｄＰＬＺＦ￣ＲＡＲα￣ｄｒｉｖｅｎｌｅｕｋｅｍｉａ[Ｊ].Ｃｌｉｎ.ＣａｎｃｅｒＲｅｓ.ꎬ２０１６ꎬ２１(１６):３６８５－３６９４.[３７]㊀ＭａＬꎬＬｉＧꎬＺｈｕＨꎬｅｔａｌ..２￣Ｍｅｔｈｏｘｙｅｓｔｒａｄｉｏｌｓｙｎｅｒｇｉｚｅｓｗｉｔｈｓｏｒａｆｅｎｉｂｔｏｓｕｐｐｒｅｓｓｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａｂｙｓｉｍｕｌｔａｎｅｏｕｓｌｙｄｙｓｒｅｇｕｌａｔｉｎｇｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ａｎｄ￣２[Ｊ].ＣａｎｃｅｒＬｅｔｔ.ꎬ２０１４ꎬ３５５(１):９６－１０５.[３８]㊀ＳｈｕｋｌａＳＫꎬＰｕｒｏｈｉｔＶꎬＭｅｈｌａＫꎬｅｔａｌ..ＭＵＣ１ａｎｄＨＩＦ￣１ａｌｐｈａｓｉｇｎａｌｉｎｇｃｒｏｓｓｔａｌｋｉｎｄｕｃｅｓａｎａｂｏｌｉｃｇｌｕｃｏｓｅｍｅｔａｂｏｌｉｓｍｔｏｉｍｐａｒｔｇｅｍｃｉｔａｂｉｎｅｒｅｓｉｓｔａｎｃｅｔｏｐａｎｃｒｅａｔｉｃｃａｎｃｅｒ[Ｊ].ＣａｎｃｅｒＣｅｌｌꎬ２０１７ꎬ３２(１):７１－８７.[３９]㊀ＹｕＴꎬＴａｎｇＢꎬＳｕｎＸ.Ｄｅｖｅｌｏｐｍｅｎｔｏｆｉｎｈｉｂｉｔｏｒｓｔａｒｇｅｔｉｎｇｈｙ￣ｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ１ａｎｄ２ｆｏｒｃａｎｃｅｒｔｈｅｒａｐｙ[Ｊ].ＹｏｎｓｅｉＭｅｄ.Ｊ.ꎬ２０１７ꎬ５８(３):４８９－４９６.[４０]㊀ＷｕＤꎬＰｏｔｌｕｒｉＮꎬＬｕＪꎬｅｔａｌ..Ｓｔｒｕｃｔｕｒａｌｉｎｔｅｇｒａｔｉｏｎｉｎｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒｓ[Ｊ].Ｎａｔｕｒｅꎬ２０１５ꎬ５２４(７５６５):３０３－３０８.[４１]㊀ＭｉｒａｎｄａＥꎬＮｏｒｄｇｒｅｎＩＫꎬＭａｌｅＡＬꎬｅｔａｌ..ＡｃｙｃｌｉｃｐｅｐｔｉｄｅｉｎｈｉｂｉｔｏｒｏｆＨＩＦ￣１ｈｅｔｅｒｏｄｉｍｅｒｉｚａｔｉｏｎｔｈａｔｉｎｈｉｂｉｔｓｈｙｐｏｘｉａｓｉｇ￣ｎａｌｉｎｇｉｎｃａｎｃｅｒｃｅｌｌｓ[Ｊ].Ｊ.Ａｍ.Ｃｈｅｍ.Ｓｏｃ.ꎬ２０１３ꎬ１３５(２８):１０４１８－１０４２５.[４２]㊀ＳｃｈｅｕｅｒｍａｎｎＴＨꎬＬｉＱꎬＭａＨＷꎬｅｔａｌ..Ａｌｌｏｓｔｅｒｉｃｉｎｈｉｂｉｔｉｏｎｏｆｈｙｐｏｘｉａｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣２ｗｉｔｈｓｍａｌｌｍｏｌｅｃｕｌｅｓ[Ｊ].Ｎａｔ.Ｃｈｅｍ.Ｂｉｏｌ.ꎬ２０１３ꎬ９(４):２７１－２７６.[４３]㊀ＳｒｉＬＭꎬＳｕｄｈａｎａＳＭ.ＭｏｌｅｃｕｌａｒｄｏｃｋｉｎｇｂａｓｅｄｓｃｒｅｅｎｉｎｇｏｆａｓｉｍｕｌａｔｅｄＨＩＦ￣１ｐｒｏｔｅｉｎｍｏｄｅｌｆｏｒｐｏｔｅｎｔｉａｌｉｎｈｉｂｉｔｏｒｓ[Ｊ].Ｂｉｏｉｎｆｏｒｍａｔｉｏｎꎬ２０１７ꎬ１３(１１):３８８－３９３.[４４]㊀ＰｏｒｔｕｇａｌＪ.ＣｈａｌｌｅｎｇｉｎｇｔｒａｎｓｃｒｉｐｔｉｏｎｂｙＤＮＡ￣ｂｉｎｄｉｎｇａｎｔｉｔｕｍｏｒｄｒｕｇｓ[Ｊ].Ｂｉｏｃｈｅｍ.Ｐｈａｒｍａｃｏｌ.ꎬ２０１８ꎬ１５５:３３６－４５. [４５]㊀ＪａｙａｔｕｎｇａＭＫꎬＴｈｏｍｐｓｏｎＳꎬＭｃｋｅｅＴＣꎬｅｔａｌ..ＩｎｈｉｂｉｔｉｏｎｏｆｔｈｅＨＩＦ１ａｌｐｈａ￣ｐ３００ｉｎｔｅｒａｃｔｉｏｎｂｙｑｕｉｎｏｎｅ￣ａｎｄｉｎｄａｎｄｉｏｎｅｄ￣ｍｅｄｉａｔｅｄｅｊｅｃｔｉｏｎｏｆｓｔｒｕｃｔｕｒａｌＺｎ(ＩＩ)[Ｊ].Ｅｕｒ.Ｊ.Ｍｅｄ.Ｃｈｅｍ.ꎬ２０１５ꎬ９４:５０９－５１６.[４６]㊀ＲｅｅｃｅＫＭꎬＲｉｃｈａｒｄｓｏｎＥＤꎬＣｏｏｋＫＭꎬｅｔａｌ..Ｅｐｉｄｉｔｈｉｏ￣ｄｉｋｅｔｏｐｉｐｅｒａｚｉｎｅｓ(ＥＴＰｓ)ｅｘｈｉｂｉｔｉｎｖｉｔｒｏａｎｔｉａｎｇｉｏｇｅｎｉｃａｎｄｉｎｖｉｖｏａｎｔｉｔｕｍｏｒａｃｔｉｖｉｔｙｂｙｄｉｓｒｕｐｔｉｎｇｔｈｅＨＩＦ￣１α/ｐ３００ｃｏｍｐｌｅｘｉｎａｐｒｅｃｌｉｎｉｃａｌｍｏｄｅｌｏｆｐｒｏｓｔａｔｅｃａｎｃｅｒ[Ｊ].Ｍｏｌ.Ｃａｎｃｅｒꎬ２０１４ꎬ１３(１):９１－１０３.[４７]㊀ＺｉｍｎａＡꎬＫｕｒｐｉｓｚＭ.Ｈｙｐｏｘｉａ￣ｉｎｄｕｃｉｂｌｅｆａｃｔｏｒ￣１ｉｎｐｈｙｓｉｏｌｏｇｉｃａｌａｎｄｐａｔｈｏｐｈｙｓｉｏｌｏｇｉｃａｌａｎｇｉｏｇｅｎｅｓｉｓ:Ａｐｐｌｉｃａｔｉｏｎｓａｎｄｔｈｅｒａｐｉｅｓ[Ｊ].Ｂｉｏｍｅｄ.Ｒｅｓ.Ｉｎｔ.ꎬ２０１５:５４９４１２. [４８]㊀ＭａｓｏｕｄＧＮꎬＷａｎｇＪꎬＣｈｅｎＪꎬｅｔａｌ..ＤｅｓｉｇｎꎬｓｙｎｔｈｅｓｉｓａｎｄｂｉｏｌｏｇｉｃａｌｅｖａｌｕａｔｉｏｎｏｆｎｏｖｅｌＨＩＦ１αｉｎｈｉｂｉｔｏｒｓ[Ｊ].ＡｎｔｉｃａｎｃｅｒＲｅｓ.ꎬ２０１５ꎬ３５(７):３８４９－３８５９.[４９]㊀ＪｉａｎｇＬꎬＳｈｉＳꎬＳｈｉＱꎬｅｔａｌ..ＳｉｍｉｌａｒｉｔｙｉｎｔｈｅｆｕｎｃｔｉｏｎｓｏｆＨＩＦ￣１ａｌｐｈａａｎｄＨＩＦ￣２ａｌｐｈａｐｒｏｔｅｉｎｓｉｎｃｅｒｖｉｃａｌｃａｎｃｅｒｃｅｌｌｓ[Ｊ].Ｏｎｃｏｌ.Ｌｅｔｔ.ꎬ２０１７ꎬ１４(５):５６４３－５６５１.[５０]㊀ＧｕａｎＺꎬＤｉｎｇＣꎬＤｕＹꎬｅｔａｌ..ＨＡＦｄｒｉｖｅｓｔｈｅｓｗｉｔｃｈｏｆＨＩＦ￣１αｔｏＨＩＦ￣２αｂｙａｃｔｉｖａｔｉｎｇｔｈｅＮＦ￣κＢｐａｔｈｗａｙꎬｌｅａｄｉｎｇｔｏｍａ￣ｌｉｇｎａｎｔｂｅｈａｖｉｏｒｏｆＴ２４ｂｌａｄｄｅｒｃａｎｃｅｒｃｅｌｌｓ[Ｊ].Ｉｎｔ.Ｊ.Ｏｎｃｏｌ.ꎬ２０１４ꎬ４４(２):３９３－４０２.[５１]㊀Ｍａｒｔíｎｅｚ￣ＳáｅｚＯꎬＢｏｒａｕＰＧꎬＡｌｏｎｓｏ￣ＧｏｒｄｏａＴꎬｅｔａｌ..Ｔａｒｇｅ￣ｔｉｎｇＨＩＦ￣２αｉｎｃｌｅａｒｃｅｌｌｒｅｎａｌｃｅｌｌｃａｒｃｉｎｏｍａ:Ａｐｒｏｍｉｓｉｎｇｔｈｅｒａｐｅｕｔｉｃｓｔｒａｔｅｇｙ[Ｊ].Ｃｒｉｔ.Ｒｅｖ.Ｏｎｃｏｌ.Ｈｅｍａｔ.ꎬ２０１７ꎬ１１１:１１７－１２３.０４３生物技术进展ＣｕｒｒｅｎｔＢｉｏｔｅｃｈｎｏｌｏｇｙ. All Rights Reserved.。