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Biomedical subjects

Linda M Hendershot

Publications and source records attributed to Linda M Hendershot.

At least 19 recordsLinked to original sources
PubMed2007

Organization of the functions and components of the endoplasmic reticulum.

The endoplasmic reticulum is the site of entry into the secretory pathway and represents a major and particularly crowded site of protein biosynthesis. In addition to the complexity of protein folding in any organelle, the ER environment poses further dangers and constraints to the process. A quality control apparatus exists to monitor the maturation of proteins in the ER. Nascent polypeptide chains are specifically prevented from traveling further along the secretory pathway until they have completed their folding or assembly. Proteins that cannot achieve a proper conformation are recognized and removed from the ER for degradation by the 26S proteasome. Finally, the homeostasis of the ER is vigilantly monitored and changes that impinge upon the proper maturation of proteins in this organelle lead to the activation of a signal transduction cascade that serves to restore balance to the ER. Recent studies suggest that some of these diverse functions may be achieved due to the organization of the ER into functional and perhaps even physical sub-domains.

Animals↗
PubMed2006

UPR activation alters chemosensitivity of tumor cells.

A growing body of literature demonstrates that the unfolded protein response (UPR) is activated in a number of tumors. Recent data suggests that the UPR may play a role in protecting transformed cells from the inadequate environment that exists prior to vascularization and therefore contribute to tumor growth and survival. In addition, data from cell culture based studies demonstrate that pharmacological activation of the UPR can alter the sensitivity of cells to chemotherapeutic agents, making them either more sensitive in some cases or more resistant in others. Thus, it will be important to understand and integrate data on the role of the UPR in tumor growth and survival with studies on the effects of UPR activation on chemosensitivity before considering therapeutic interventions into this signaling pathway.

Antineoplastic Agents↗
PubMed2005

Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy.

SIL1 (also called BAP) acts as a nucleotide exchange factor for the Hsp70 chaperone BiP (also called GRP78), which is a key regulator of the main functions of the endoplasmic reticulum. We found nine distinct mutations that would disrupt the SIL1 protein in individuals with Marinesco-Sjögren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. Identification of SIL1 mutations implicates Marinesco-Sjögren syndrome as a disease of endoplasmic reticulum dysfunction and suggests a role for this organelle in multisystem disorders.

Adolescent↗
PubMed2005

Activation of the unfolded protein response is necessary and sufficient for reducing topoisomerase IIalpha protein levels and decreasing sensitivity to topoisomerase-targeted drugs.

A wide range of chemotherapeutic agents has been identified that are active against solid tumors. However, resistance remains an important obstacle to the development of curative regimens. Whereas much attention has been paid to acquired drug resistance, a variety of physiological pathways also have been described that reduce the sensitivity of previously untreated tumors to cytotoxic antitumor agents. Treatment of cells with pharmacological agents that alter the environment of the endoplasmic reticulum (ER) and activate the unfolded protein response (UPR) can render cells resistant to topoisomerase II poisons. We describe experiments showing that activation of the mammalian ER stress response is both necessary and sufficient to decrease topoisomerase IIalpha protein levels and to render cells resistant to etoposide, a topoisomerase II-targeting drug. This is not caused by the elevated levels of BiP that are a hallmark of this response, because a cell line that has been engineered to overexpress BiP does not show increased resistance to etoposide. The UPR was shown to be required for altered drug sensitivity, because the BiP-overexpressing cell line, which is unable to activate the UPR, did not show decreased topoisomerase II levels or increased resistance to etoposide in response to stress conditions. The transient overexpression of an unfolded protein activated the UPR and led to the concomitant loss of topoisomerase IIalpha protein from the cells, demonstrating that UPR activation is sufficient for the changes in topoisomerase II levels that had been observed previously with pharmacological induction of the UPR.

Animals↗
PubMed2005

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum.

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.

Adenosine Diphosphate↗
PubMed2005

Building an antibody factory: a job for the unfolded protein response.

Plasma cells are highly specialized, terminally differentiated secretory cells that produce tremendous quantities of a single product, the antibody molecule. In differentiating from a quiescent B cell, the plasma cell must undergo a dramatic architectural metamorphosis. This process entails augmenting the secretory organelles and the proteins that populate them, upregulating their energy and translation potential, and increasing the quality control system to do the job. This transformation is accomplished by an interplay between B lineage-specific transcriptional programs that control plasma cell differentiation and an unfolded protein response.

Animals↗
PubMed2005

ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum.

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain. A highly positively charged nonapeptide within this domain is necessary and sufficient for binding to ribosomes. Binding of ERj1p to ribosomes involves the 28S ribosomal RNA and occurs at the tunnel exit. Additionally, ERj1p has a dual regulatory role in gene expression: ERj1p inhibits translation in the absence of BiP, and another charged oligopeptide within the cytosolic domain of ERj1p mediates binding of the nuclear import factor importin beta and import into the nucleus, thereby paving the way for subsequent action on genomic DNA.

Animals↗
PubMed2004

ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates.

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

Adenosine Triphosphatases↗
PubMed2004

GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.

Animals↗
PubMed2004

Herp is dually regulated by both the endoplasmic reticulum stress-specific branch of the unfolded protein response and a branch that is shared with other cellular stress pathways.

The mammalian unfolded protein response (UPR) includes two major branches: one(s) specific to ER stress (Ire1/XBP-1 and ATF6-dependent), and one(s) shared by other cellular stresses (PERK/eIF-2alpha phosphorylation-dependent). Here, we demonstrate that the ER-localized protein Herp represents a second target, in addition to CHOP, that is dually regulated by both the shared and the ER stress-specific branches during UPR activation. For the first time, we are able to assess the contribution of each branch of the UPR in the induction of these targets. We demonstrate that activation of the shared branch of the UPR alone was sufficient to induce Herp and CHOP. ATF4 was not required during ER stress when both branches were used but did contribute significantly to their induction. Conversely, stresses that activated only the shared branch of the UPR were completely dependent on ATF4 for CHOP and Herp induction. Thus, the shared and the ER stress-specific branches of the UPR diverge to regulate two groups of targets, one that is ATF6 and Ire1/XBP-1-dependent, which includes BiP and XBP-1, and another that is eIF-2alpha kinase-dependent, which includes ATF4 and GADD34. The two branches also converge to maximally up-regulate targets like Herp and CHOP. Finally, our studies reveal that a PERK-dependent target other than ATF4 is contributing to the cross-talk between the two branches of the UPR that has previously been demonstrated.

Activating Transcription Factor 4↗
PubMed2004

ER chaperone functions during normal and stress conditions.

Nearly all resident proteins of the organelles along the secretory pathway, as well as proteins that are expressed at the cell surface or secreted from the cell, are first co-translationally translocated into the lumen of the endoplasmic reticulum (ER) as unfolded polypeptide chains. Immediately after entering the ER, they are often modified with N-linked glycans, are folded into the appropriate secondary and tertiary structures, which are stabilized by disulfide bonds, and finally in many cases are assembled into multimeric complexes. These processes are aided and monitored by ER chaperones and folding enzymes. When cells experience conditions that alter the ER environment, protein folding can be dramatically affected and can lead to the accumulation of unfolded proteins in this organelle. This in turn activates a signaling response, which is shared among all eukaryotic organisms, termed the unfolded protein response (UPR). The hallmark of this response is the coordinate transcriptional up-regulation of ER chaperones and folding enzymes. A major role for the increased levels of chaperones and folding enzymes during conditions of ER stress is to provide the same functions they carry out during normal physiological conditions. This includes preventing unfolded and incompletely folded proteins from aggregating and promoting the proper folding and assembly of proteins in the ER. During conditions of ER stress, many proteins are unable to fold properly and the requirements for chaperones are therefore increased. However, more recently it has become clear that some ER chaperones are also involved in signaling the ER stress response, targeting misfolded proteins for degradation and perhaps even shutting down the UPR when the stress subsides. In addition, during some normal physiological conditions, like plasma cell differentiation where there is an increased demand in the secretory capacity of B cells, the levels of various ER chaperones are also up-regulated via at least part of the UPR pathway. In order to discuss these various functions of ER chaperones, we will begin with the roles of ER chaperones and folding enzymes during normal physiological conditions and then discuss their roles during ER stress.

Animals↗
PubMed2004

The role of the unfolded protein response in tumour development: friend or foe?

Having accumulated mutations that overcome cell-cycle and apoptotic checkpoints, the main obstacle to survival faced by a cancer cell is the restricted supply of nutrients and oxygen. These conditions impinge on protein folding in the endoplasmic reticulum and activate a largely cytoprotective signalling pathway called the unfolded protein response. Prolonged activation of this response can, however, terminate in apoptosis. Recent delineation of the components of this response, coupled with several clinical studies, indicate that it is uniquely poised to have a role in regulating the balance between cancer cell death, dormancy and aggressive growth, as well as altering the sensitivity of solid tumours to chemotherapeutic agents.

Antineoplastic Agents↗
PubMed2004

The ER function BiP is a master regulator of ER function.

The endoplasmic reticulum (ER) is a command center of the cell that is second only to the nucleus in terms of the breadth of its influence on other organelles and activities. It is a major site of protein synthesis, contains the cellular calcium stores that are an essential component of many signaling pathways, and is the proximal site of a signal transduction cascade that responds to cellular stress conditions and serves to maintain homeostasis of the cell. All eucaryotic cells possess an ER, which can comprise nearly 50% of the membranes of a cell. Its functions can be divided into those that occur on the cytosolic side of the membrane (where protein translation and signal transduction cascades occur) and the luminal space (where most other ER functions take place). Our studies during the past several years have revealed that the ER molecular chaperone BiP is a master regulator of ER function. It is responsible for maintaining the permeability barrier of the ER during protein translocation, directing protein folding and assembly, targeting misfolded proteins for retrograde translocation so they can be degraded by the proteasome, contributing to ER calcium stores, and sensing conditions of stress in this organelle, to activate the mammalian unfolded protein response.

Adaptation, Physiological↗
PubMed2003

Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress.

Transient protein synthesis inhibition is an important protective mechanism used by cells during various stress conditions including endoplasmic reticulum (ER) stress. This response centers on the phosphorylation state of eukaryotic initiation factor (eIF)-2 alpha, which is induced by kinases like protein kinase R-like ER kinase (PERK) and GCN2 to suppress translation and is later reversed so translation resumes. GADD34 was recently identified as the factor that activates the type 1 protein serine/threonine phosphatase (PP1), which dephosphorylates eIF-2 alpha during cellular stresses. Our study delineates a negative feedback regulatory loop in which the eIF-2 alpha-controlled inhibition of protein translation leads to GADD34 induction, which promotes translational recovery. We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eIF-2 alpha-mediated translational block, is required for the transactivation of the GADD34 promoter in response to ER stress and amino acid deprivation. ATF4 directly binds to and trans-activates a conserved ATF site in the GADD34 promoter during ER stress. Examination of ATF4-/- MEFs revealed an absence of GADD34 induction, prolonged eIF-2 alpha phosphorylation, delayed protein synthesis recovery, and diminished translational up-regulation of BiP during ER stress. These studies demonstrate the essential role of GADD34 in the resumption of protein synthesis, define the pathway for its induction, and reveal that cytoprotective unfolded protein response targets like BiP are sensitive to the eIF-2 alpha-mediated block in translation.

Activating Transcription Factor 4↗
PubMed2002

BAP, a mammalian BiP-associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP.

We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

Adenosine Triphosphatases↗
PubMed2002

Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response.

CHOP is a non-ER localized transcription factor that is induced by a variety of adverse physiological conditions including ER stress. Accumulation of unfolded proteins in the ER activates an unfolded protein response pathway that targets both ER resident chaperones (e.g. BiP) and CHOP. Hence, it is unclear if CHOP induction during ER stress occurs through the ER stress response element that is conserved in both CHOP and ER chaperone promoters, or through a separate regulatory pathway conserved among different CHOP inducing cellular stress conditions. We identified a bona fide ER stress element in the hamster CHOP promoter and found that similar transcription complexes containing NF-Y bound to both the CHOP and BiP ER stress response elements. In addition, we demonstrated for the first time the importance of the C/EBP-ATF composite site for CHOP regulation during ER stress. Activation of the ER transmembrane eIF2alpha kinase, PERK, induced ATF4 protein expression, direct binding to the composite site in CHOP promoter, and as a consequence, CHOP protein induction. We propose that this eIF2alpha-kinase/ATF4/C/EBP-ATF composite site pathway is conserved for CHOP regulation during various cellular stress conditions including ER stress. Our data indicate that both the ERSE and the PERK-ATF4 pathways converge on the CHOP promoter during ER stress and provide insights into the similarities and differences between CHOP and ER chaperone expression during normal and stress conditions.

Animals↗