Proteostasis Regulators Research Articles

Lacidipine Remodels Protein Folding and Ca 2+ Homeostasis in Gaucher's Disease Fibroblasts: A Mechanism to Rescue Mutant Glucocerebrosidase

The hallmark of Gaucher's disease cellular pathogenesis is the lysosomal accumulation of glucosylceramide, which is caused by misfolding of mutated glucocerebrosidase (GC) and loss of lysosomal GC activity, and leads to depletion of [Ca(2+)](ER). We demonstrate that modulation of Ca(2+) homeostasis and enhancement of the cellular folding capacity synergize to rescue the folding of mutated GC variants. Lacidipine, an L-type Ca(2+) channel blocker that also inhibits [Ca(2+)](ER) efflux, enhances folding, trafficking, and activity of degradation-prone GC variants. Lacidipine remodels mutated GC proteostasis by simultaneously activating a series of distinct molecular mechanisms, namely modulation of Ca(2+) homeostasis, upregulation of the ER chaperone BiP, and moderate induction of the unfolded protein response. However, unlike previously reported proteostasis regulators, lacidipine treatment is not cytotoxic but prevents apoptosis induction typically associated with sustained activation of the unfolded protein response.

Proteostasis: a new therapeutic paradigm for pulmonary disease.

Among lung pathologies, α1AT, chronic obstructive pulmonary disease (COPD), emphysema, and asthma are diseases triggered by local environmental stress in the airway that we refer to herein collectively as airway stress diseases (ASDs). A deficiency of α-1-antitrypsin (α1AT) is an inherited genetic disorder that is a consequence of the misfolding of α1AT during protein synthesis in liver hepatocytes, reducing secretion to the plasma and delivery to the lung. Deficiency of α1AT in the lung triggers a similar pathological phenotype to other ASDs. Moreover, the loss of α1AT in the lung is a well-known environmental risk factor for COPD/emphysema. To date there are no effective therapeutic approaches to address ASDs, which reflects a general lack of understanding of their cellular basis. Herein, we propose that ASDs are disorders of proteostasis. That is, they are initiated and propagated by a common theme-a challenge to protein folding capacity maintained by the proteostasis network (PN) (see Balch et al., Science 2008;319:916-919). The PN is a network of chaperones and degradative components that generates and manages protein folding pathways responsible for normal human physiology. In ASD, we suggest that the PN system fails to respond to the increased burden of unfolded proteins due to genetic and environmental stresses, thus triggering pulmonary pathophysiology. We introduce the enabling concept of proteostasis regulators (PRs), small molecules that regulate signaling pathways that control the composition and activity of PN components, as a new and general approach for therapeutic management of ASDs.

Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function

Neurofibromatosis type 2 (NF2) is a multiple neoplasia syndrome and is caused by a mutation of the NF2 tumor suppressor gene that encodes for the tumor suppressor protein merlin. Biallelic NF2 gene inactivation results in the development of central nervous system tumors, including schwannomas, meningiomas, ependymomas, and astrocytomas. Although a wide variety of missense germline mutations in the coding sequences of the NF2 gene can cause loss of merlin function, the mechanism of this functional loss is unknown. To gain insight into the mechanisms underlying loss of merlin function in NF2, we investigated mutated merlin homeostasis and function in NF2-associated tumors and cell lines. Quantitative protein and RT-PCR analysis revealed that whereas merlin protein expression was significantly reduced in NF2-associated tumors, mRNA expression levels were unchanged. Transfection of genetic constructs of common NF2 missense mutations into NF2 gene-deficient meningioma cell lines revealed that merlin loss of function is due to a reduction in mutant protein half-life and increased protein degradation. Transfection analysis also demonstrated that recovery of tumor suppressor protein function is possible, indicating that these mutants maintain intrinsic functional capacity. Further, increased expression of mutant protein is possible after treatment with specific proteostasis regulators, implicating protein quality control systems in the degradative fate of mutant tumor suppressor proteins. These findings provide direct insight into protein function and tumorigenesis in NF2 and indicate a unique treatment paradigm for this disorder.

Assessing Proteostasis and Proteasome Stress In Light Chain Amyloidosis

AbstractAbstract 3992Recently, proteasome inhibitors (PI) proved powerful against multiple myeloma (MM), the neoplastic transformation of plasma cells. The balance between proteasome expression and degradative workload (mainly contributed by protein synthesis) proved a crucial determinant of apoptotic sensitivity of MM cells to proteasome inhibition (Bianchi et al, Blood 2009). Light chain amyloidosis (AL) is a plasma cell dyscrasia caused by a bone marrow plasma cell clone synthesizing structurally unstable, misfolded, monoclonal immunoglobulin (Ig) light chains, which polymerize into amyloid fibrils. Interestingly, AL is proving even more sensitive than MM to PI in clinical trials with unprecedented response rates (>80%) rapidly achieved in previously untreated patients (Kastritis et al, J Clin Oncol 2010), raising the question as to whether, and if so why, AL cells are intrinsically more sensitive than MM to PI. We hypothesized that AL cells suffer from intense proteasome stress linked to the synthesis of the misfolded Ig light chain, thereby facing constitutive proteotoxicity.To test this hypothesis, we set out to optimize purification of primary bone marrow plasma cells from AL patients, and determine: intrinsic sensitivity to the PI bortezomib (by FACS); proteasome activity (by fluorogenic assays); accumulation of ubiquitinated (Ub) proteins and Ig light chain (by immunofluorescence).Our ex vivo studies demonstrated twofold higher PI sensitivity in AL plasma cells as compared to primary MM cells (EC50 in 24 hr apoptosis assays: AL, 8.3 ± 2.2 nM; MM, 15.1 ± 3.0 nM). We also found that, similar to MM cells, proteasome activity of primary AL plasma cells varies greatly among different patients (5.2 ± 3.6 nM substrate specifically cleaved by the chymotryptic β-peptidase activity per cell per min). Furthermore, accumulation of Ub proteins strongly correlates with light chain content, suggesting a crucial role for paraprotein synthesis and/or retention on proteasome stress. Interestingly, unlike MM cells, we failed to detect a clear correlation between proteasome activity and ex vivo assessed PI sensitivity, possibly due to intracellular toxicity of the misfolded light chain. The resulting hypothesis that different mutations could result in different intrinsic proteotoxicity in AL cells is currently being tested.In conclusion, our integrated approach indicates that AL cells are intrinsically more sensitive to PI than MM cells, providing a potential explanation for the excellent clinical responses. Moreover, we established a technological platform to investigate proteostasis and proteotoxic stress in primary AL cells. This strategy may help investigate the efficacy of proteostasis regulators on plasma cell dyscrasias, including MM, and identify molecular markers of clinical use to predict disease severity and response to therapy. Disclosures:No relevant conflicts of interest to declare.

Biological and Chemical Approaches to Diseases of Proteostasis Deficiency

Many diseases appear to be caused by the misregulation of protein maintenance. Such diseases of protein homeostasis, or "proteostasis," include loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer's, Parkinson's, and Huntington's disease). Proteostasis is maintained by the proteostasis network, which comprises pathways that control protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. The decreased ability of the proteostasis network to cope with inherited misfolding-prone proteins, aging, and/or metabolic/environmental stress appears to trigger or exacerbate proteostasis diseases. Herein, we review recent evidence supporting the principle that proteostasis is influenced both by an adjustable proteostasis network capacity and protein folding energetics, which together determine the balance between folding efficiency, misfolding, protein degradation, and aggregation. We review how small molecules can enhance proteostasis by binding to and stabilizing specific proteins (pharmacologic chaperones) or by increasing the proteostasis network capacity (proteostasis regulators). We propose that such therapeutic strategies, including combination therapies, represent a new approach for treating a range of diverse human maladies.

Chemical and Biological Approaches Synergize to Ameliorate Protein-Folding Diseases

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.

Adapting Proteostasis for Disease Intervention

The protein components of eukaryotic cells face acute and chronic challenges to their integrity. Eukaryotic protein homeostasis, or proteostasis, enables healthy cell and organismal development and aging and protects against disease. Here, we describe the proteostasis network, a set of interacting activities that maintain the health of proteome and the organism. Deficiencies in proteostasis lead to many metabolic, oncological, neurodegenerative, and cardiovascular disorders. Small-molecule or biological proteostasis regulators that manipulate the concentration, conformation, quaternary structure, and/or the location of protein(s) have the potential to ameliorate some of the most challenging diseases of our era.