glomerular visceral epithelial cells ( gecs ; podocytes ) play a key role in the maintenance of glomerular permselectivity [ 14 ] . under normal conditions , podocytes are in contact with extracellular matrix , and there appears to be little turnover of podocytes , as these cells are highly differentiated (  terminally differentiated  ) . gecs form a tight network of interdigitating foot processes , which are bridged by filtration - slit diaphragms , and permselectivity of the glomerular capillary wall is dependent on the maintenance of appropriate structure of podocytes and of their foot processes . the complex cellular architecture of the podocyte is maintained by an organization of actin filaments in the cytoplasm . proteins including nephrin , neph1 , fat , podocin , cd2ap and zo-1 are found within or near the slit diaphragm , and nephrin , neph family proteins , and cadherins form complexes with scaffolding proteins , cd2ap and zo-1 [ 2 , 4 ] . these connect the slit - diaphragm protein complex with actin filaments , which are joined by -actinin-4 proteins . acquired gec injury is frequently associated with effacement of the foot processes , disruption of the filtration - slit diaphragms , and proteinuria [ 1 , 2 ] . moreover , heritable mutations in several gec structural proteins alter podocyte ultrastructure and cause heritable proteinuria , implying that impairment of the slit diaphragm or cytoskeletal structure underlies proteinuria [ 4 , 5 ] . focal segmental glomerulosclerosis ( fsgs ) is an important cause of proteinuria and nephrotic syndrome in humans . in recent years , evidence has accumulated to support the view that the early pathogenesis of fsgs is associated with gec injury , leading to apoptosis , detachment of gecs from extracellular matrix , and  podocytopenia  , which is followed by glomerulosclerosis [ 13 , 68 ] . acquired gec injury may be triggered by immunological factors ( e.g. , t cell or other factors affecting permeability ) , oxidants , human immunodeficiency virus , toxins , and other substances . in addition , fsgs may be associated with heritable mutations in several distinct proteins that play key roles in maintaining gec ultrastructure , including podocin , -actinin-4 , transient receptor potential cation channel , subfamily c , member 6 ( trpc6 ) , inverted formin 2 and others . in this paper , we focus on mutations in -actinin-4 , which are associated with an autosomal dominant late - onset fsgs in humans . we discuss the structure and function of -actinin-4 , and we review mechanisms by which mutations in -actinin-4 may lead to podocyte injury and fsgs . there are four -actinin genes ( actn1 - 4 ) that encode highly homologous proteins ( -actinin-1 , 2 , 3 and 4 ) , which form ~100  kda head - to - tail homodimers . -actinins-2 and -3 are ca - insensitive muscle -actinins expressed in z - discs of striated muscle cells , while -actinins-1 and -4 are widely distributed , nonmuscle isoforms . all family members are actin crosslinking proteins that share a number of structural features and regulatory regions . these include a central rod with four spectrin - like repeats , two n - terminal calponin - homology domains ( ch1 and ch2 ) , which contain three actin - binding sites ( abs1 - 3 ) , a c - terminal calmodulin - like domain , containing two putative ef hands , and a phosphoinositide - binding plextrin homology domain . the spectrin - like repeats facilitate homodimerization and may confer a degree of flexibility to the -actinin molecule , allowing it to resist mechanical strain . for its interaction with actin filaments , the ch1 and ch2 domains adopt a closed conformation , where abs 2 and 3 are exposed , while abs1 remains buried and inaccessible to f - actin . this association with actin is highly dynamic and can be controlled by the regulatory regions along the -actinin sequence . all -actinin isoforms contain two putative ef - hand motifs at the c - terminus of each monomer , but only isoforms 1 and 4 demonstrate sensitivity to ca [ 15 , 16 ] . for these nonmuscle isoforms , binding of ca is believed to reduce the affinity of -actinin for actin , providing a mechanism for -actinin - dependent cytoskeletal remodeling . additionally , extensive biochemical studies on the effect of phosphoinositide binding to -actinin-1 have been performed . binding of phosphatidylinositol ( 3,4,5)-trisphosphate ( pip3 ) , and to a lesser extent phosphatidylinositol ( 4,5)-bisphosphate ( pip2 ) , decreases the association between -actinin-1 and f - actin , suggesting that phosphoinositides mediate cytoskeletal remodeling [ 17 , 18 ] . for -actinin-4 , in contrast to findings for -actinin-1 , our studies showed that phosphoinositides increase the interaction of -actinin-4 with f - actin . tyrosine phosphorylation at position 12 was found to be dependent on focal adhesion kinase ( fak ) , supporting a role for -actinin in cell adhesion . phosphorylation of -actinin-1 reduced its association with f - actin in vitro , suggesting that -actinin mediates fak - dependent cytoskeletal remodeling . the amino acid sequence of -actinin-4 reveals an analogous tyrosine residue at position 32 , suggesting that phosphorylation may also regulate the binding of -actinin-4 to f - actin . however , we were unable to demonstrate fak - dependent tyrosine phosphorylation of -actinin-4 ( unpublished observations ) . expression of these isoforms at the cytoplasmic face of several types of cellular interaction sites , including focal adhesions , adherens junctions , and hemidesmosomes suggests a degree of versatility . the first -actinin binding partners identified were the  subunits of integrins and intercellular adhesion molecule-1 . since -actinins also bind to various regulatory molecules , -actinin may also serve as a scaffolding protein to integrate signaling molecules at various adhesion sites . -actinin-1 may link membrane proteins such as talin , vinculin , and -integrins with the cortical actin cytoskeleton [ 10 , 2225 ] . similarly , the tight junction protein , magi-1 , interacts with the c - terminus of -actinin-4 , providing a physical link from the cell periphery to the cytoskeleton . the list of interacting proteins is likely to grow and will provide a better understanding of the diverse functions of -actinins in nonmuscle tissues . however , the best defined function of -actinin-4 is to crosslink / bundle actin filaments , and it may enhance cell motility and elicit tumor - suppressor activity .   intriguingly , -actinin-4 is reported to shuttle between the cytoplasm and the nucleus . cytoplasmic localization was associated with an infiltrative phenotype and correlated significantly with a poorer prognosis in cases of breast cancer . in this context , the relevance of nuclear -actinin-4 remains unknown , although it has been suggested that nuclear translocation may attenuate metastatic potential . the nuclear localization of -actinin-4 suggests a role for -actinin-4 in gene expression . -actinins-1 and -4 were identified as histone deacetylase 7 ( hdac7)-interacting proteins , and the interaction domain was mapped to the c - terminus of -actinin 4 . hdac7 can participate in multiple cellular processes , including the regulation of myocyte enhancer factor-2- ( mef2- ) mediated transcription . a point mutation in hdac7 disrupted its association with -actinin-4 and mef2 , implying that -actinin-4 and mef2 binding sites overlap . overexpression of -actinin-4 also potentiated estrogen receptor -mediated transcription by counteracting a negative regulatory effect of hdac7 , while knockdown of -actinin-4 decreased expression of estrogen receptor  target genes and affected proliferation of cultured breast cancer cells . another study demonstrated that -actinin-4 colocalized along actin stress fibers and in membrane lamellae with nuclear factor-b ( nf-b ) . incubation of cells with epidermal growth factor or tumor necrosis factor- induced translocation of -actinin-4 and the p65 subunit of nf-b into the nucleus . as nf-b regulates transcription of a large number of genes in response to diverse stimuli , the study further supports a regulatory role for -actinin-4 in transcription . human gecs express only -actinin-4 ( although mouse gecs also express -actinin-1 ) [ 10 , 30 ] . immunoelectron microscopy studies showed that -actinin localizes to contractile microfilaments within podocyte foot processes [ 31 , 32 ] . in cultured mouse gecs , -actinin-4 was found along actin stress fibers , focal adhesions , and within the cortical actin network at the cell periphery ( figure 1 ) . this specific subcellular localization of -actinin-4 facilitates its regulation of adhesion and cytoskeletal dynamics [ 19 , 33 , 36 ] . interestingly , -actinin-4 was also present in the nucleus of some rat gecs [ 34 , 35 ] , in keeping with earlier reports in other cell lines . normally , -actinin-4 may be required for integrin - dependent adhesion of gecs . indeed , knockout of -actinin-4 in mice resulted in reduced glomerular podocyte number , accompanied by the appearance of urinary wilm 's tumor-1 ( wt-1 ) , a podocyte biomarker . such podocyte loss is consistent with defective glomerular basement membrane adhesion , as tunel assays failed to detect any apoptotic cells in -actinin-4 null glomeruli . the severity of the resulting phenotype seen in -actinin-4 null mice ( i.e. , glomerular disease , renal failure , and early death ) indicates that -actinin-4-mediated podocyte adhesion is critical for filtration barrier function [ 30 , 37 , 38 ] . on the other hand , transgenic podocyte overexpression of wild - type -actinin-4 in mice these findings suggest that podocytes tolerate a wide range of -actinin-4 expression , but that a minimum threshold level is essential for normal gec adhesion to the glomerular basement membrane through its interaction with integrins . deficiency of -actinin-4 leading to human disease is not described , but several point mutations or single amino acid deletions in -actinin-4 are found in heritable forms of human fsgs . most mutations in actn4-associated fsgs families congregate at the ch1-ch2 interface [ 9 , 40 ] . an example is the autosomal dominant k255e mutation ( k256e is the mouse mutant corresponding to human k255e ) . such mutants show increased binding to actin filaments , compared with the wild - type protein [ 30 , 41 , 42 ] . interestingly , -actinin-4 k256e is insensitive to regulation by ca and phosphoinositides ( pip2 and pip3 ) , suggesting that its gain of affinity for f - actin blunts its responsiveness to regulatory factors . expression of an -actinin-4 k256e transgene in mouse podocytes in vivo ( under the control of the nephrin promoter ) resulted in development of proteinuric fsgs . in addition , homozygous ( but not heterozygous )  knock - in  of the mutant -actinin-4 allele into the actn4 locus in mice induced proteinuria , confirming the pathogenic potential of -actinin-4 mutations . the underlying biochemical mechanism , whereby fsgs - associated actn4 mutations enhance affinity for actin , was uncovered by pollak 's group . they showed that disease - causing mutations disable an important intramolecular hinge that normally holds ch1 and ch2 in a  closed  conformation . the mutant protein adopts an open conformation forcing an interaction of all three actin - binding sites with the actin filament , thereby increasing the binding affinity by lowering its rate of dissociation from actin . although -actinin-4 is widely expressed , human podocytes appear to be selectively vulnerable to mutations in -actinin-4 ( including k256e and other analogous mutants ) , perhaps because podocytes are  terminally differentiated  cells with limited regenerative capacity . the downstream cellular mechanism(s ) by which mutant -actinin-4 leads to podocyte injury and fsgs is poorly understood . one possibility is that injury is secondary to alterations in the mechanical properties of the podocyte via increased affinity of mutant -actinin-4 for f - actin ( figure 2 ) . gecs that express -actinin-4 k256e show defective spreading and motility ( figure 1 ) . moreover , exposure of -actinin-4 k256e expressing gecs to cyclical equibiaxial stretch , an in vitro mimic of the mechanical forces due to glomerular capillary pressure , significantly reduced cell surface area and caused retraction of cellular processes . lastly , the enhanced association with f - actin alters the subcellular localization of -actinin-4 , restricting its presence at the cell periphery ( figure 1 ) , and potentially altering its capacity to interact with various binding partners [ 19 , 33 ] . such abnormalities could , at least in part , be attributable to cytoskeletal disruption , as well as loss of integrin engagement , effectively yielding a phenotype similar to that of the -actinin-4 null mouse [ 36 , 43 ] . since the actin cytoskeleton is a key component contributing to the unique morphological characteristics of the podocyte foot processes , as well as being connected to both slit diaphragm and adhesion complexes , altered -actinin-4 biology must have important implications for podocyte health . an alternate and/or parallel mechanism , which we also consider in this paper , is that mutant -actinin-4 induces proteotoxicity in podocytes ( i.e. , an impairment of podocyte function caused by misfolding of a protein ) , ultimately leading to apoptosis ( figure 2 ) . consistent with this notion , -actinin-4 k256e forms actin - associated aggregates in cultured gecs and in podocytes of both homozygous k256e  knock - in  mice and humans with actn4-associated fsgs [ 30 , 33 , 35 ] . such misfolded / aggregated protein may be associated with the activation of stress pathways in podocytes ( see below ) . prior to discussing the proteotoxic potential of mutant -actinin-4 , this section provides a brief overview of the ubiquitin - proteasome system and er stress . the ubiquitin - proteasome system plays a key role in regulating the half - life of short - lived cellular proteins , and in selective degradation of damaged or abnormal proteins [ 45 , 46 ] . the proteasome degrades aberrant cytoplasmic or cytoskeletal proteins , and misfolded er proteins , which are retrotranslocated selectively to the cytoplasm . the latter process is known as er - associated degradation ( erad ) [ 47 , 48 ] . ubiquitin - proteasome pathway - mediated protein degradation involves tagging of the substrate by covalent attachment of ubiquitin molecules via a three - step reaction , and degradation of the tagged protein by the 26s proteasome complex . ubiquitinated proteins typically undergo efficient degradation by the proteasome , but sometimes , large amounts of misfolded proteins are not degraded effectively , and may form covalently - linked aggregates . such misfolded proteins and/or aggregates may impair the function of the proteasome and lead to the activation of stress pathways and cytotoxicity . secretory , luminal , and membrane proteins normally attain their correctly folded conformation in the er via er - resident chaperones . to rescue misfolded proteins , the er has in place quality control machinery , including the unfolded protein response ( upr ) [ 4953 ] , and erad [ 47 , 48 ] . in the upr , accumulation of misfolded proteins in the er results in the activation of three er  sensors  . activating transcription factor-6 moves from the er to the golgi , where it is cleaved by proteases . the cleaved cytosolic fragment translocates to the nucleus to activate transcription of er chaperones , for example , the glucose - regulated proteins ( grp ) , grp94 and bip ( grp78 ) , and others . in parallel , inositol requiring-1 activates its endoribonuclease activity , cleaving x - box binding protein-1 mrna and changing the reading frame to yield a potent transcriptional activator . normally , er chaperones assist in posttranslational processing of proteins and in their exit from the er , and may complex with defective proteins to target them for degradation . during stress , induction of er chaperones may enhance the protein folding capacity , and limit accumulation of abnormal proteins . misfolded proteins in the er also activate perk ( pkr - like er kinase ) , which then phosphorylates the eukaryotic translation initiation factor-2 subunit ( eif2 ) . this process reduces initiation aug codon recognition , and the general rate of translation is blunted ( which decreases the protein load on a damaged er ) . the upr allows cells to recover from stress , and may be protective to additional insults , but substantial / prolonged er stress may lead to apoptosis . for example , certain mrnas may be translated preferentially after eif2 is phosphorylated . among these is activating transcription factor-4 , which induces expression of several genes , including chop ( c / ebp homologous protein-10 ; also known as gadd153 ) , a gene closely associated with apoptosis and/or growth arrest [ 49 , 51 ] . apoptosis may also result from activation of caspase-12 or protein kinases [ 49 , 51 ] . impairment of the ubiquitin - proteasome system can be associated with exacerbation of er stress [ 49 , 54 ] , perhaps by interference with erad . the potential for mutant -actinin-4 to impair podocyte function is suggested by the characteristics of this mutant protein to form microaggregates , undergo ubiquitination , impair the ubiquitin - proteasome system , enhance er stress , and enhance apoptosis ( figure 2 ) . density - gradient centrifugation of the k255e mutant -actinin-4 showed abnormal sedimentation , suggesting that the mutant protein forms high molecular mass aggregates . in keeping with this result , expression of -actinin-4 k255e ( and other fsgs - inducing mutants ) , but not wild - type -actinin-4 in cultured mouse gecs resulted in formation of aggregates , as visualized by fluorescence microscopy [ 30 , 33 , 42 , 43 ] . analogous results were obtained in rat gecs stably transfected with -actinin-4 k256e , where in some cells , the mutant ( but not the wild type ) protein formed aggregates . unlike the wild - type protein , the mutant was not present in the nucleus of rat gecs [ 34 , 35 ] . in addition , -actinin-4 k255e formed aggregates in podocytes of homozygous k255e  knock - in  mice and humans with actn4-associated fsgs . misfolded / aggregated proteins may result in the activation of stress pathways .   in pulse - chase metabolic labeling experiments , mutant -actinin-4 was degraded more rapidly , compared with the wild - type protein , with the mutant showing a half - life of ~15  h , and the wild type of over 30  h . consistent with this result , after stable transfection in rat gecs or transient transfection in cos cells ( a monkey kidney cell line , which allows for high levels of protein expression ) , the level of -actinin-4 k256e protein was considerably lower , compared with the wild - type protein ( figure 3 ) [ 34 , 35 ] . together , these results suggested that -actinin-4 k256e may undergo ubiquitination prior to degradation via the ubiquitin - proteasome pathway . indeed , in transiently transfected cos cells , mutant -actinin-4 was polyubiquitinated , whereas the wild - type protein was not ( figure 3 ) . treatment of gecs that express -actinin-4 k256e with proteasome inhibitors enhanced expression of -actinin-4 k256e , in keeping with ubiquitination and degradation of the mutant protein by the proteasome [ 30 , 35 ] . -actinin-4 k256e and wild - type proteins were transiently overexpressed in cos cells to study their effects on the ubiquitin - proteasome system . the function of the ubiquitin - proteasome system was monitored in viable cells by use of a reporter consisting of a short degron , cl1 , fused to the c - terminus of green fluorescent protein ( gfp ) . this gfp proteasome reporter is rapidly degraded when ubiquitin - proteasome function is normal , whereas impairment of the ubiquitin - proteasome system , for example , via enhanced flux of a mutant / aggregated protein will reduce degradation of gfp , resulting in an increased level of gfp expression . in cos cells transfected with wild - type -actinin-4 , expression of gfp declined significantly , in keeping with proteasomal degradation of gfp ( figure 3 ) . in contrast , expression of -actinin-4 k256e retarded the degradation of gfp , implying that the ubiquitin - proteasome system was impaired in the presence of mutant -actinin-4 . -actinin-4 is a cytosolic / cytoskeletal protein that normally would not enter the er to undergo posttranslational modification . nonetheless , compared with -actinin-4 wild type , transient transfection of -actinin-4 k256e in cos cells enhanced the upr , as evidenced by increased expression of the er chaperone , grp94 , and the proapoptotic gene , chop ( figure 3 ) . in gecs , expression of the k256e mutant or wild type -actinin-4 ( by stable transfection ) did not increase expression of the er chaperone , bip , or chop , compared with parental ( untransfected ) cells . this difference between cos cells and gecs may be due to lower expression of stably transfected proteins in the gecs . however , after incubation of gecs with tunicamycin ( a drug that blocks n - linked glycosylation and causes an accumulation of misfolded proteins in the er ) , the gecs stably transfected with -actinin-4 k256e showed increases in bip and chop that significantly exceeded the increases seen in cells expressing the wild - type protein , as well as the increases in parental gecs . the effect of -actinin-4 k256e on the induction of the upr by tunicamycin was particularly robust , given that expression of the mutant was substantially lower than the wild - type protein . thus , although stable expression of -actinin-4 k256e in gecs did not induce the upr directly , the mutant appeared to adversely affect the integrity of the er , which may render these cells more susceptible to additional stress and induction of proapoptotic genes . together , these results are in keeping with the view that exacerbation of er stress may be secondary to impairment of the ubiquitin - proteasome system [ 49 , 54 ] . stable expression of -actinin-4 k256e in gecs led to a reduction in cell number , as well as increased apoptosis and caspase-3 activity , compared with gecs that stably express -actinin-4 wild type , or parental ( untransfected ) gecs . these changes in cell number and apoptosis in the presence of -actinin-4 k256e occurred despite the significantly lower expression level of the mutant , compared with the wild - type -actinin-4 , highlighting the potential detrimental consequences of this mutation . in cos cells , -actinin-4 k256e had only a slight effect on apoptosis and cell number , suggesting that this cell type may be more resistant to cytotoxicity . nevertheless , -actinin-4 k256e markedly exacerbated apoptosis and reduced cell number in the context of mild proteasome inhibition . this result supports the view that apoptosis induced by mutant -actinin-4 may be associated with an impairment in proteasome function . the effects of -actinin-4 k256e on the upr ( discussed above ) are based on studies in cultured cell lines , but importantly , analogous changes also occurred in vivo . as stated above , transgenic mice that express an -actinin-4 k256e transgene in podocytes develop proteinuria and fsgs . expression of the er chaperones , bip and grp94 , eif2 phosphorylation , as well as expression of the proapoptotic protein , chop , were increased in glomeruli of transgenic mice , compared with control . based on these results , it is reasonable to propose that in the -actinin-4 k256e model of fsgs , there is pronounced er stress , which may be contributing , at least in part , to gec apoptosis . the maintenance of a highly dynamic actin - based cytoskeleton is critically important to podocyte morphology and function . microfilaments in the foot processes tether the actin cytoskeleton to the slit diaphragm and adhesion complexes , while forming the architectural infrastructure for the foot processes . -actinin-4 provides structural support to these microfilaments via its crosslinking and bundling activities , while linking them to components of the slit diaphragm and sites of adhesion . the gain affinity mutations in fsgs - associated -actinin-4 substantially alter the properties of the actin cytoskeleton , rendering it more rigid and less dynamic . therefore , the underlying pathogenesis of actn4-associated podocyte injury , glomerular filtration barrier dysfunction and the appearance of fsgs lesions are at least partly attributable to an aberrantly high interaction of -actinin-4 with f - actin and its impact upon the cytoskeleton . in addition , the enhanced actin--actinin-4 interaction generates misfolded protein / aggregates , which could provide a parallel mechanism of podocyte dysfunction . as discussed above , misfolded proteins may induce dysfunction of the ubiquitin - proteasome system , that is , the misfolded proteins  choke  or this process may enhance proapoptotic stress in cellular compartments , including the er . in addition , since ubiquitination regulates many essential cellular processes , including normal protein degradation , cell cycle , transcription , dna repair , and protein trafficking , a disrupted ubiquitin - proteasome system may have broader adverse consequences for cell function . thus , the pathogenesis of fsgs associated with -actinin-4 k256e may resemble processes in certain age - related or neurodegenerative diseases , where signs of er stress , ups dysfunction , and protein misfolding are observed [ 30 , 45 , 54 , 5658 ] . for example , in huntington 's disease , the expansion of a glutamine stretch within the n - terminal region of huntingtin gene generates a protein with severe neurotoxic properties . expression of mutant huntingtin leads to a pronounced defect in erad , and upr activation was noted in postmortem huntington 's disease brains . familial amyotrophic lateral sclerosis has been linked to mutations in the gene encoding superoxide dismutase-1 , and these mutations induce misfolding and aggregation of this protein , which is believed to contribute to neuronal dysfunction and death . activation of the upr is observed in mutant superoxide dismutase-1 transgenic mice , and increased levels of er stress markers , as well as wild - type superoxide dismutase-1 aggregates have been reported in spinal cord tissue of sporadic amyotrophic lateral sclerosis . modulation of er stress pathways protected mice with experimental amyotrophic lateral sclerosis from disease progression . other examples of neurodegenerative diseases associated with protein misfolding / aggregation and er stress are alzheimer 's , parkinson 's , and prion diseases . protein misfolding and ups dysfunction also appears to play a role in desmin - related cardiomyopathies , which result in congestive heart failure . by analogy to experimental neurodegenerative disease models , one must , however , be cautious in extrapolating the cell culture events , which are based on overexpression and a relatively brief experimental time frame , to a disease process that in humans takes many years to become established . finally , the shuttling of the wild type , but not mutant -actinin-4 between the cytoplasm and the nucleus , as well as the potential for disruption of gene regulation in the presence of mutant -actinin-4 , will require additional consideration as a potential mechanism of podocyte injury in fsgs .
focal segmental glomerulosclerosis ( fsgs ) is an important cause of proteinuria and nephrotic syndrome in humans . 
 the pathogenesis of fsgs may be associated with glomerular visceral epithelial cell ( gec ; podocyte ) injury , leading to apoptosis , detachment , and 
 podocytopenia  , followed by glomerulosclerosis . 
 mutations in -actinin-4 are associated with fsgs in humans . in cultured gecs , 
 -actinin-4 mediates adhesion and cytoskeletal dynamics . 
 fsgs - associated -actinin-4 mutants show increased binding to actin filaments , compared with the wild - type protein . 
 expression of an -actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric fsgs . 
 gecs that express mutant -actinin-4 show defective spreading and motility , and such abnormalities could alter the mechanical properties of the podocyte , contribute to cytoskeletal disruption , and lead to injury . the potential for mutant -actinin-4 to injure podocytes 
 is also suggested by the characteristics of this mutant protein to form microaggregates , undergo ubiquitination , impair the ubiquitin - proteasome system , enhance endoplasmic reticulum stress , and exacerbate apoptosis .