The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for ...degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70. Surprisingly, the CHIP-TPR interacts with determinants within both the Hsc70-lid subdomain and the C-terminal PTIEEVD motif of the tail, exhibiting an atypical mode of interaction between chaperones and TPR domains. We demonstrate that the interaction between CHIP and the Hsc70-lid subdomain is required for proper ubiquitination of Hsp70/Hsc70 or Hsp70/Hsc70-bound substrate proteins. Posttranslational modifications of the Hsc70 lid and tail disrupt key contacts with the CHIP-TPR and may regulate CHIP-mediated ubiquitination. Our study shows how CHIP docks onto Hsp70/Hsc70 and defines a bipartite mode of interaction between TPR domains and their binding partners.
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•Hsc70/Hsp70 engage in novel bipartite binding mode with CHIP•Hsp70-lid interaction with CHIP is required for ubiquitination of Hsp70 clients•TPR:lid-tail structure allows modeling of full-length Hsp70:CHIP complexes•Phosphorylation or methylation of Hsp70-lid residues regulate interaction with CHIP
Zhang et al. report a novel interaction between Hsp70 and CHIP. This interaction allows for full-length models of the CHIP/Hsp70 complex to be assembled, predicts how Hsp70 posttranslational modifications regulate ubiquitination, and suggests a new mode of chaperone/cochaperone interactions.
Brain tumor cells invade adjacent normal brain along white matter (WM) bundles of axons. We therefore hypothesized that the location of tumor intersecting WM tracts would be associated with differing ...survival. This study introduces a method, voxel-wise survival analysis (VSA), to determine the relationship between the location of brain tumor intersecting WM tracts and patient prognosis. 113 primary glioblastoma (GBM) patients were retrospectively analyzed for this study. Patient specific tumor location, defined by contrast-enhancement, was combined with diffusion tensor imaging derived tractography to determine the location of axons intersecting tumor enhancement (AXITEs). VSA was then used to determine the relationship between the AXITE location and patient survival. Tumors intersecting the right anterior thalamic radiation (ATR), right inferior fronto-occipital fasciculus (IFOF), right and left cortico-spinal tract (CST), and corpus callosum (CC) were associated with decreased overall survival. Tumors intersecting the CST, body of the CC, right ATR, posterior IFOF, and inferior longitudinal fasciculus are associated with decreased progression-free survival (PFS), while tumors intersecting the right genu of the CC and anterior IFOF are associated with increased PFS. Patients with tumors intersecting the ATR, IFOF, CST, or CC had significantly improved survival prognosis if they were additionally treated with bevacizumab. This study demonstrates the usefulness of VSA by locating AXITEs associated with poor prognosis in GBM patients. This information should be included in patient-physician conversations, therapeutic strategy, and clinical trial design.
The social amoeba
's proteome contains a vast array of simple sequence repeats, providing a unique model to investigate proteostasis. Upon conditions of cellular stress,
undergoes a developmental ...process, transitioning from a unicellular amoeba to a multicellular fruiting body. Little is known about how proteostasis is maintained during
's developmental process. Here, we have identified a novel α-crystallin domain-containing protein, heat shock protein 48 (HSP48), that is upregulated during
development. HSP48 functions in part by forming a biomolecular condensate via its highly positively charged intrinsically disordered carboxy terminus. In addition to HSP48, the highly negatively charged primordial chaperone polyphosphate is also upregulated during
development, and polyphosphate functions to stabilize HSP48. Upon germination, levels of both HSP48 and polyphosphate dramatically decrease, consistent with a role for HSP48 and polyphosphate during development. Together, our data demonstrate that HSP48 is strongly induced during
development. We also demonstrate that HSP48 forms a biomolecular condensate and that polyphosphate is necessary to stabilize the HSP48 biomolecular condensate.
During cellular stress, many microbes undergo a transition to a dormant state. This includes the social amoeba
that transitions from a unicellular amoeba to a multicellular fruiting body upon starvation. In this work, we identify heat shock protein 48 (HSP48) as a chaperone that is induced during development. We also show that HSP48 forms a biomolecular condensate and is stabilized by polyphosphate. The findings here identify
as a novel microbe to investigate protein quality control pathways during the transition to dormancy.
Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations ...in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.
Although there is convergent evidence for blood-brain barrier (BBB) dysfunction and peripheral inflammation in schizophrenia (SZ) and bipolar disorder (BD), it is unknown whether BBB deficits are ...intrinsic to brain microvascular endothelial cells (BMECs) or arise via effects of peripheral inflammatory cytokines. We examined BMEC function using stem cell-based models to identify cellular and molecular deficits associated with BBB dysfunction in SZ and BD. Induced pluripotent stem cells (iPSCs) from 4 SZ, 4 psychotic BD and 4 healthy control (HC) subjects were differentiated into BMEC-"like" cells. Gene expression and protein levels of tight junction proteins were assessed. Transendothelial electrical resistance (TEER) and permeability were assayed to evaluate BBB function. Cytokine levels were measured from conditioned media. BMECs derived from human iPSCs in SZ and BD did not show differences in BBB integrity or permeability compared to HC BMECs. Outlier analysis using TEER revealed a BBB-deficit (n = 3) and non-deficit (n = 5) group in SZ and BD lines. Stratification based on BBB function in SZ and BD patients identified a BBB-deficit subtype with reduced barrier function, tendency for increased permeability to smaller molecules, and decreased claudin-5 (CLDN5) levels. BMECs from the BBB-deficit group show increased matrix metallopeptidase 1 (MMP1) activity, which correlated with reduced CLDN5 and worse BBB function, and was improved by tumor necrosis factor α (TNFα) and MMP1 inhibition. These results show potential deficits in BMEC-like cells in psychotic disorders that result in BBB disruption and further identify TNFα and MMP1 as promising targets for ameliorating BBB deficits.
Cognitive impairment is the strongest predictor of functional outcomes in schizophrenia and is hypothesized to result from synaptic dysfunction. However, targeting synaptic plasticity and cognitive ...deficits in patients remains a significant clinical challenge. A comprehensive understanding of synaptic plasticity and the molecular basis of learning and memory in a disease context can provide specific targets for the development of novel therapeutics targeting cognitive impairments in schizophrenia. Here, we describe the role of synaptic plasticity in cognition, summarize evidence for synaptic dysfunction in schizophrenia and demonstrate the use of patient derived induced-pluripotent stem cells for studying synaptic plasticity in vitro. Lastly, we discuss current advances and future technologies for bridging basic science research of synaptic dysfunction with clinical and translational research that can be used to predict treatment response and develop novel therapeutics.
The expression, misfolding, and aggregation of long repetitive amino acid tracts are a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral ...sclerosis/frontotemporal dementia, fragile X tremor ataxia syndrome, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, and human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.
Background: The Dictyostelium proteome is predicted to encode a vast amount of homopolymeric amino acid tracts, including long polyglutamine tracts.
Results: Proteins with long polyglutamine tracts are soluble in Dictyostelium.
Conclusion: Polyglutamine proteins do not form aggregates in Dictyostelium under normal growth conditions.
Significance:Dictyostelium possess properties that suppress protein aggregation that may be co-opted to address polyglutamine toxicity in human disease.
Protein aggregation is one of the hallmarks of many neurodegenerative diseases. While protein aggregation is a heavily studied aspect of neurodegenerative disease, methods of detection vary from one ...model system to another. Induced pluripotent stem cells (iPSCs) present an opportunity to model disease using patient-specific cells. However, iPSC-derived neurons are fetal-like in maturity, making it a challenge to detect key features such as protein aggregation that are often exacerbated with age. Nevertheless, we have previously found abnormal soluble and insoluble protein burden in motor neurons generated from amyotrophic lateral sclerosis (ALS) iPSCs, though protein aggregation has not been readily detected in iPSC-derived neurons from other neurodegenerative diseases. Therefore, here we present an ultracentrifugation method that detects insoluble protein species in various models of neurodegenerative disease, including Huntington's disease, Alzheimer's disease, and ALS. This method is able to detect soluble, insoluble, and SDS-resistant species in iPSC-derived neurons and is designed to be flexible for optimal detection of various aggregation-prone proteins.
Age-related neurodegenerative diseases are an incurable class of diseases that afflict an increasing percentage of the population. While the pathophysiology differs, protein aggregation is a hallmark ...of a number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and the nine polyglutamine (polyQ) diseases. The polyQ diseases are caused by the expansion of a CAG repeat within the coding region of specific genes, resulting in the expression of an aggregation-prone, polyQ-expanded protein. Expression of a polyQ-expanded tract results in protein aggregation in both human disease as well as many model organisms tested, including yeast, worms, flies, mice, rats, and human cells. Interestingly, the social amoeba, Dictyostelium discoideum, contains more simple sequence repeats than any other genome sequenced, resulting in a large number of homopolymeric repeats. Among the most abundant are polyQ tracts, with some tracts reaching lengths that would be considered pathogenic in humans. To investigate this, I first showed that Dictyostelium expresses proteins with long polyQ tracts and is able to maintain their solubility. Moreover, overexpression of a human polyQ-expanded protein did not result in protein aggregation, suggesting Dictyostelium is highly resistant to polyQ aggregation. To identify the mechanism by which Dictyostelium prevents polyQ aggregation, I performed a forward genetic screen that led to the discovery of a novel molecular chaperone that conveys resistance to polyQ aggregation, which we termed serine-rich chaperone protein 1 (SRCP1). SRCP1 inhibits the aggregation of polyQ-expanded proteins via its C-terminus and allows for the degradation of aggregation-prone polyQ proteins via the proteasome. These results identify a novel type of molecular chaperone and yields insight into how nature has dealt with protein aggregation. Upon conditions of cellular stress, Dictyostelium also undergoes a developmental process, transitioning from a unicellular amoeba to a multicellular fruiting body. In addition to analyzing Dictyostelium’s protein quality control network at the unicellular state, I also sought out to identify mechanisms by which Dictyostelium maintain proteostasis during development. Here, I identified an α-crystallin domain containing protein, heat shock protein 48 (HSP48), that is greatly upregulated in Dictyostelium development. I further showed HSP48 phase separates to form a biomolecular condensate via its highly charged, intrinsically disordered C-terminus. Interestingly, the chemical chaperone polyphosphate is also greatly upregulated in Dictyostelium development and functions to stabilize HSP48 protein levels. These data suggest that Dictyostelium development may also serve as a unique system for studying phase separation as well as protein quality control pathways. Overall, my thesis work with SRCP1 and HSP48 establish Dictyostelium as a unique model system to study protein quality control pathways and provide a promising venue for the development of novel therapeutics to combat protein aggregation.
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