Distinct patterns of proteostasis network gene expression are associated with different prognoses in melanoma patients.

The proteostasis network (PN) is a collection of protein folding and degradation pathways that spans cellular compartments and acts to preserve the integrity of the proteome. The differential expression of PN genes is a hallmark of many cancers, and the inhibition of protein quality control factors is an effective way to slow cancer cell growth. However, little is known about how the expression of PN genes differs between patients and how this impacts survival outcomes. To address this, we applied unbiased hierarchical clustering to gene expression data obtained from primary and metastatic cutaneous melanoma (CM) samples and found that two distinct groups of individuals emerge across each sample type. These patient groups are distinguished by the differential expression of genes encoding ATP-dependent and ATP-independent chaperones, and proteasomal subunits. Differences in PN gene expression were associated with increased levels of the transcription factors, MEF2A, SP4, ZFX, CREB1 and ATF2, as well as markedly different survival outcomes. However, surprisingly, similar PN alterations in primary and metastatic samples were associated with discordant survival outcomes in patients. Our findings reveal that the expression of PN genes demarcates CM patients and highlights several new proteostasis sub-networks that could be targeted for more effective suppression of CM within specific individuals.

Chaperone therapy for lysosomal and non-lysosomal protein misfolding diseases.

Chaperone therapy was introduced first as a new molecular therapeutic approach to lysosomal diseases. In a recent article, I reviewed the development of chaperone therapy mainly for lysosomal diseases. Then, more data have been collected particularly on non-lysosomal protein misfolding diseases. In this short review, I propose the concept of chaperone therapy to be classified into two different therapeutic approaches, for pH-dependent lysosomal, and pH-independent non-lysosomal protein misfolding diseases. The concept of lysosomal chaperone therapy is well established, but the non-lysosomal chaperone therapy is heterogeneous and to be investigated further for various individual diseases. As a whole, these two-types of new molecular therapeutic approaches will make an impact on the treatment of a wide range of pathological conditions caused by protein misfolding, not necessarily lysosomal but also many non-lysosomal diseases caused by gene mutations, metabolic diseases, malignancy, infectious diseases, and aging. The concept will open a completely new aspect of protein therapy in future.

ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration.

Copper is a transition metal essential for human life. Its homeostasis is regulated in the liver, which delivers copper to the whole body and excretes its excess outside the organism in the feces through the bile. These functions are regulated within hepatocytes, and the ATP7B copper transporter is central to making the switch between copper use and excretion. In Wilson disease, the gene coding for ATP7B is mutated, leading to copper overload, firstly, in the liver and the brain. To better understand the role of ATP7B in hepatocytes and to provide a smart tool for the development of novel therapies against Wilson disease, we used the CrispR/Cas9 tool to generate hepatocyte cell lines with the abolished expression of ATP7B. These cell lines revealed that ATP7B plays a major role at low copper concentrations starting in the micromolar range. Moreover, metal stress markers are induced at lower copper concentrations compared to parental cells, while redox stress remains not activated. As shown recently, the main drawback induced by copper exposure is protein unfolding that is drastically exacerbated in ATP7B-deficient cells. Our data enabled us to propose that the zinc finger domain of DNAJ-A1 would serve as a sensor of Cu stress. Therefore, these Wilson-like hepatocytes are of high interest to explore in more detail the role of ATP7B.

Secretagogin is a Ca2+-dependent stress-responsive chaperone that may also play a role in aggregation-based proteinopathies.

Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca2+-binding protein that plays a regulatory role in the release of several hormones. SCGN is expressed largely in pancreatic ß-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.

Misfolding-induced chronic pancreatitis in CPA1 N256K mutant mice is unaffected by global deletion of Ddit3/Chop.

Genetic mutations in pancreatic digestive enzymes may cause protein misfolding, endoplasmic reticulum (ER) stress and chronic pancreatitis. The CPA1 N256K mouse model carries the human p.N256K carboxypeptidase A1 (CPA1) mutation, a classic example of a pancreatitis-associated misfolding variant. CPA1 N256K mice develop spontaneous, progressive chronic pancreatitis with moderate acinar atrophy, acinar-to-ductal metaplasia, fibrosis, and macrophage infiltration. Upregulation of the ER-stress associated pro-apoptotic transcription factor Ddit3/Chop mRNA was observed in the pancreas of CPA1 N256K mice suggesting that acinar cell death might be mediated through this mechanism. Here, we crossed the CPA1 N256K strain with mice containing a global deletion of the Ddit3/Chop gene (Ddit3-KO mice) and evaluated the effect of DDIT3/CHOP deficiency on the course of chronic pancreatitis. Surprisingly, CPA1 N256K x Ddit3-KO mice developed chronic pancreatitis with a similar time course and features as the CPA1 N256K parent strain. In contrast, Ddit3-KO mice showed no pancreas pathology. The observations indicate that DDIT3/CHOP plays no significant role in the development of misfolding-induced chronic pancreatitis in CPA1 N256K mice and this transcription factor is not a viable target for therapeutic intervention in this disease.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.

Expression of Mutant Ubiquitin and Proteostasis Impairment in Kii Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex Brains.

Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder that is endemic to the Kii peninsula of Japan. The disorder is clinically characterized by a variable combination of parkinsonism, dementia, and motor neuron symptoms. Despite extensive investigations, the etiology and pathogenesis of ALS/PDC remain unclear. At the neuropathological level, Kii ALS/PDC is characterized by neuronal loss and tau-dominant polyproteinopathy. Here, we report the accumulation of several proteins involved in protein homeostasis pathways, that is, the ubiquitin-proteasome system and the autophagy-lysosome pathway, in postmortem brain tissue from a number of Kii ALS/PDC cases (n = 4). Of particular interest is the presence of a mutant ubiquitin protein (UBB+1), which is indicative of disrupted ubiquitin homeostasis. The findings suggest that abnormal protein aggregation is linked to impaired protein homeostasis pathways in Kii ALS/PDC.

How Does the Ribosome Fold the Proteome?

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.

Alpha 1-Antitrypsin Deficiency: A Disorder of Proteostasis-Mediated Protein Folding and Trafficking Pathways.

Human cells express large amounts of different proteins continuously that must fold into well-defined structures that need to remain correctly folded and assemble in order to ensure their cellular and biological functions. The integrity of this protein balance/homeostasis, also named proteostasis, is maintained by the proteostasis network (PN). This integrated biological system, which comprises about 2000 proteins (chaperones, folding enzymes, degradation components), control and coordinate protein synthesis folding and localization, conformational maintenance, and degradation. This network is particularly challenged by mutations such as those found in genetic diseases, because of the inability of an altered peptide sequence to properly engage PN components that trigger misfolding and loss of function. Thus, deletions found in the ΔF508 variant of the Cystic Fibrosis (CF) transmembrane regulator (CFTR) triggering CF or missense mutations found in the Z variant of Alpha 1-Antitrypsin deficiency (AATD), leading to lung and liver diseases, can accelerate misfolding and/or generate aggregates. Conversely to CF variants, for which three correctors are already approved (ivacaftor, lumacaftor/ivacaftor, and most recently tezacaftor/ivacaftor), there are limited therapeutic options for AATD. Therefore, a more detailed understanding of the PN components governing AAT variant biogenesis and their manipulation by pharmacological intervention could delay, or even better, avoid the onset of AATD-related pathologies.

Quantitating the epigenetic transformation contributing to cholesterol homeostasis using Gaussian process.

To understand the impact of epigenetics on human misfolding disease, we apply Gaussian-process regression (GPR) based machine learning (ML) (GPR-ML) through variation spatial profiling (VSP). VSP generates population-based matrices describing the spatial covariance (SCV) relationships that link genetic diversity to fitness of the individual in response to histone deacetylases inhibitors (HDACi). Niemann-Pick C1 (NPC1) is a Mendelian disorder caused by >300 variants in the NPC1 gene that disrupt cholesterol homeostasis leading to the rapid onset and progression of neurodegenerative disease. We determine the sequence-to-function-to-structure relationships of the NPC1 polypeptide fold required for membrane trafficking and generation of a tunnel that mediates cholesterol flux in late endosomal/lysosomal (LE/Ly) compartments. HDACi treatment reveals unanticipated epigenomic plasticity in SCV relationships that restore NPC1 functionality. GPR-ML based matrices capture the epigenetic processes impacting information flow through central dogma, providing a framework for quantifying the effect of the environment on the healthspan of the individual.

Association of Aggresomes with Survival Outcomes in Pediatric Medulloblastoma.

Aggresomes are inclusion bodies for misfolded/aggregated proteins. Despite the role of misfolded/aggregated proteins in neurological disorders, their role in cancer pathogenesis is poorly defined. In the current study we aimed to investigate whether aggresomes-positivity could be used to improve the disease subclassification and prognosis prediction of pediatric medulloblastoma. Ninety three pediatric medulloblastoma tumor samples were retrospectively stratified into three molecular subgroups; WNT, SHH and non-WNT/non-SHH, using immunohistochemistry and Multiplex Ligation Probe Amplification. Formation of aggresomes were detected using immunohistochemistry. Overall survival (OS) and event-free survival (EFS) were determined according to risk stratification criteria. Multivariate Cox regression analyses were carried out to exclude confounders. Aggresomes formation was detected in 63.4% (n = 59/93) of samples. Aggresomes were non-randomly distributed among different molecular subgroups (P = 0.00002). Multivariate Cox model identified aggresomes' percentage at ≥20% to be significantly correlated with patient outcome in both OS (HR = 3.419; 95% CI, 1.30-8.93; P = 0.01) and EFS (HR = 3; 95% CI, 1.19-7.53; P = 0.02). The presence of aggresomes in ≥20% of the tumor identified poor responders in standard risk patients; OS (P = 0.02) and EFS (P = 0.06), and significantly correlated with poor outcome in non-WNT/non-SHH molecular subgroup; OS (P = 0.0002) and EFS (P = 0.0004).

Factors in the disease severity of ATP1A3 mutations: Impairment, misfolding, and allele competition.

Dominant mutations of ATP1A3, a neuronal Na,K-ATPase α subunit isoform, cause neurological disorders with an exceptionally wide range of severity. Several new mutations and their phenotypes are reported here (p.Asp366His, p.Asp742Tyr, p.Asp743His, p.Leu924Pro, and a VUS, p.Arg463Cys). Mutations associated with mild or severe phenotypes [rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), or early infantile epileptic encephalopathy (EIEE)] were expressed in HEK-293 cells. Paradoxically, the severity of human symptoms did not correlate with whether there was enough residual activity to support cell survival. We hypothesized that distinct cellular consequences may result not only from pump inactivation but also from protein misfolding. Biosynthesis was investigated in four tetracycline-inducible isogenic cell lines representing different human phenotypes. Two cell biological complications were found. First, there was impaired trafficking of αß complex to Golgi apparatus and plasma membrane, as well as changes in cell morphology, for two mutations that produced microcephaly or regions of brain atrophy in patients. Second, there was competition between exogenous mutant ATP1A3 (α3) and endogenous ATP1A1 (α1) so that their sum was constant. This predicts that in patients, the ratio of normal to mutant ATP1A3 proteins will vary when misfolding occurs. At the two extremes, the results suggest that a heterozygous mutation that only impairs Na,K-ATPase activity will produce relatively mild disease, while one that activates the unfolded protein response could produce severe disease and may result in death of neurons independently of ion pump inactivation.

In Vitro Monitoring Conformational Changes of Polypeptide Monolayers Using Infrared Plasmonic Nanoantennas.

Proteins and peptides play a predominant role in biochemical reactions of living cells. In these complex environments, not only the constitution of the molecules but also their three-dimensional configuration defines their functionality. This so-called secondary structure of proteins is crucial for understanding their function in living matter. Misfolding, for example, is suspected as the cause of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Ultimately, it is necessary to study a single protein and its folding dynamics. Here, we report a first step in this direction, namely ultrasensitive detection and discrimination of in vitro polypeptide folding and unfolding processes using resonant plasmonic nanoantennas for surface-enhanced vibrational spectroscopy. We utilize poly-l-lysine as a model system which has been functionalized on the gold surface. By in vitro infrared spectroscopy of a single molecular monolayer at the amide I vibrations we directly monitor the reversible conformational changes between α-helix and ß-sheet states induced by controlled external chemical stimuli. Our scheme in combination with advanced positioning of the peptides and proteins and more brilliant light sources is highly promising for ultrasensitive in vitro studies down to the single protein level.

Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation.

Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligomers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70-Bag3 complex therefore functions as an important signaling node that senses proteotoxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation.

FUS(1-359) transgenic mice as a model of ALS: pathophysiological and molecular aspects of the proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120 days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.

A case of tubulinopathy presenting with porencephaly caused by a novel missense mutation in the TUBA1A gene.

BACKGROUND: Tubulinopathies include a wide spectrum of disorders ranging from abnormal ocular movement to severe brain malformations, and typically present as diffuse agyria or perisylvian pachygyria with microcephaly, agenesis of the corpus callosum, and cerebellar hypoplasia. They are caused by the dysfunction of tubulins encoded by tubulin-related genes, and the TUBA1A gene encoding alpha-1A tubulin is most frequently responsible for this clinical entity. Porencephaly is relatively rare among patients with the TUBA1A mutations. Mild case of tubulinopathy associated with porencephaly caused by a novel TUBA1A mutation. CASE REPORT: The patient, a 10-month-old girl, presented with gross motor delay at 4 months of age and convulsions at 7 months of age. Brain magnetic resonance imaging showed porencephaly, occipital polymicrogyria, hypoplasia of the corpus callosum, volume loss of the white matter, dysgenesis of anterior limbs of internal capsules, non-separative basal ganglia, cerebellar hypoplasia, and dysplastic brainstem. We identified a novel de novo heterozygous missense mutation in the TUBA1A gene, c.381C > A (p.Asp127Glu), by whole-exome sequencing. DISCUSSION: Microtubules composed of tubulins regulate not only neuronal migration but also cell division or axon guidance. Accordingly, tubulinopathy affects the cortical lamination, brain size, callosal formation, and white matter as seen in the present case. In contrast to the previously reported cases, the present case showed milder cortical dysgenesis with a rare manifestation of porencephaly. The genotype-phenotype correlation is still unclear, and this study expands the phenotypic range of tubulinopathy.

X-Linked Spinal and Bulbar Muscular Atrophy: From Clinical Genetic Features and Molecular Pathology to Mechanisms Underlying Disease Toxicity.

Spinal and Bulbar Muscular Atrophy (SBMA) is an inherited neuromuscular disorder caused by a CAG-polyglutamine (polyQ) repeat expansion in the androgen receptor (AR) gene. Unlike other polyQ diseases, where the function of the native causative protein is unknown, the biology of AR is well understood, and this knowledge has informed our understanding of how native AR function interfaces with polyQ-AR dysfunction. Furthermore, ligand-dependent activation of AR has been linked to SBMA disease pathogenesis, and has led to a thorough study of androgen-mediated effects on polyQ-AR stability, degradation, and post-translational modifications, as well as their roles in the disease process. Transcriptional dysregulation, proteostasis dysfunction, and mitochondrial abnormalities are central to polyQ-AR neurotoxicity, most likely via a 'change-of-function' mechanism. Intriguingly, recent work has demonstrated a principal role for skeletal muscle in SBMA disease pathogenesis, indicating that polyQ-AR toxicity initiates in skeletal muscle and results in secondary motor neuron demise. The existence of robust animal models for SBMA has permitted a variety of preclinical trials, driven by recent discoveries of altered cellular processes, and some of this preclinical work has led to human clinical trials. In this chapter, we review SBMA clinical features and disease biology, discuss our current understanding of the cellular and molecular basis of SBMA pathogenesis, and highlight ongoing efforts toward therapy development.

A systematic atlas of chaperome deregulation topologies across the human cancer landscape.

Proteome balance is safeguarded by the proteostasis network (PN), an intricately regulated network of conserved processes that evolved to maintain native function of the diverse ensemble of protein species, ensuring cellular and organismal health. Proteostasis imbalances and collapse are implicated in a spectrum of human diseases, from neurodegeneration to cancer. The characteristics of PN disease alterations however have not been assessed in a systematic way. Since the chaperome is among the central components of the PN, we focused on the chaperome in our study by utilizing a curated functional ontology of the human chaperome that we connect in a high-confidence physical protein-protein interaction network. Challenged by the lack of a systems-level understanding of proteostasis alterations in the heterogeneous spectrum of human cancers, we assessed gene expression across more than 10,000 patient biopsies covering 22 solid cancers. We derived a novel customized Meta-PCA dimension reduction approach yielding M-scores as quantitative indicators of disease expression changes to condense the complexity of cancer transcriptomics datasets into quantitative functional network topographies. We confirm upregulation of the HSP90 family and also highlight HSP60s, Prefoldins, HSP100s, ER- and mitochondria-specific chaperones as pan-cancer enriched. Our analysis also reveals a surprisingly consistent strong downregulation of small heat shock proteins (sHSPs) and we stratify two cancer groups based on the preferential upregulation of ATP-dependent chaperones. Strikingly, our analyses highlight similarities between stem cell and cancer proteostasis, and diametrically opposed chaperome deregulation between cancers and neurodegenerative diseases. We developed a web-based Proteostasis Profiler tool (Pro2) enabling intuitive analysis and visual exploration of proteostasis disease alterations using gene expression data. Our study showcases a comprehensive profiling of chaperome shifts in human cancers and sets the stage for a systematic global analysis of PN alterations across the human diseasome towards novel hypotheses for therapeutic network re-adjustment in proteostasis disorders.