Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation

Espinoza, Mateo F.; Nguyen, Khanh K.; Sycks, Melody M.; Lyu, Ziqi; Quanrud, Guy M; Montoya, Maureen R; Genereux, Joseph C.

doi:10.1016/j.jbc.2022.102597

Cited by 6 publications

(12 citation statements)

References 126 publications

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“…The heterotrimeric SEC61 complex has a key role in translocation across the ER membrane for many proteins of the secretory pathway [8]. To assess the impact of R236C for protein translocation, the concentration of two proteins, VEGF and TTR, known to be dependent on SEC61 [28, 29] was determined in cell culture supernatants. The level of VEGF was decreased in UCi R236C and CPLC R236C relative to controls (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Weiand,

Sandfort,

Nadzemova

et al. 2023

Preprint

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Background & objectivesSEC61A1 encodes a central protein of the mammalian translocon machinery residing in the ER. Mutations ofSEC61A1are implicated to result in various severe diseases. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD), which originates from defects of cholangiocytes in the liver. The molecular phenotype of R236C was assessed.MethodsTwo cellular platforms were established from the same cell source of an ADPLD patient by different methodology. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to several analyses of molecular pathways that were previously associated with development of polycystic disease.ResultsUCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis.ConclusionAlthough the grade of aberrant cellular functions differed between the two platforms, overall the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways that cause ADPLD. Our approach may be valuable to decipher the molecular pathways of other rare genetic diseases using patient-specific cell models.

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Section: Resultsmentioning

confidence: 99%

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Weiand,

Sandfort,

Nadzemova

et al. 2023

Preprint

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“… Information on HSP70 localization have been obtained from Kampinga HH et al [ 12 ], Vega VL et al [ 22 ], Gehrmann M et al [ 25 ], Daugaard M et al [ 32 ], Radons J [ 33 ], Tavaria M et al [ 34 ], Mizzen LA et al [ 35 ], Hu G et al [ 36 ], Otterson GA et al [ 37 ], Vostakolaei MA et al [ 38 ], Espinoza MF et al [ 39 ], Wan T et al [ 40 ], and (accessed on 25 February 2023). …”

Section: Figurementioning

confidence: 99%

Is It Still Possible to Think about HSP70 as a Therapeutic Target in Onco-Hematological Diseases?

et al. 2023

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The search for molecules to be targeted that are involved in apoptosis resistance/increased survival and pathogenesis of onco-hematological malignancies is ongoing since these diseases are still not completely understood. Over the years, a good candidate has been identified in the Heat Shock Protein of 70kDa (HSP70), a molecule defined as “the most cytoprotective protein ever been described”. HSP70 is induced in response to a wide variety of physiological and environmental insults, allowing cells to survive lethal conditions. This molecular chaperone has been detected and studied in almost all the onco-hematological diseases and is also correlated to poor prognosis and resistance to therapy. In this review, we give an overview of the discoveries that have led us to consider HSP70 as a therapeutic target for mono- or combination-therapies in acute and chronic leukemias, multiple myeloma and different types of lymphomas. In this excursus, we will also consider HSP70 partners, such as its transcription factor HSF1 or its co-chaperones whose druggability could indirectly affect HSP70. Finally, we will try to answer the question asked in the title of this review considering that, despite the effort made by research in this field, HSP70 inhibitors never reached the clinic.

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“…After cellular attachment, transfected cells were treated with corresponding drugs by changing media, as summarized in Table S1. On the second day posttransfection, dimethyl sulfoxide (DMSO, as vehicle, tissue culture grade, Corning) or biotin-phenol (BP, 500 μM, from 0.5 M stock in DMSO, prepared in lab as described 26 ) were added to the cells through conditioned media containing residual drugs from initial treatment. Cells were incubated at 37°C for 30 min.…”

Section: Drug Treatment and Proximity Labelingmentioning

confidence: 99%

“…Proximity labeling has emerged as a technique of choice for characterizing subcellular proteomes 19 and protein trafficking [20][21][22][23][24] . We recently demonstrated that proximity labeling is an effective method for identifying mistargeting of secretory proteins 25,26 . In this approach, N-terminal FLAG-tagged APEX2 with a nuclear export signal (NES) 27,28 is expressed and localized in the cytosol ( cyt APEX).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Lyu

Genereux

2023

Preprint

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Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin (FLAGTTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

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Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation

Cited by 6 publications

References 126 publications

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Molecular determinants of SEC61A1 mutant R236C in two patient-derived cellular platforms

Is It Still Possible to Think about HSP70 as a Therapeutic Target in Onco-Hematological Diseases?

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

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