Proteostasis

The human B cell survival factor, B cell activation factor (BAFF), and its closely related homologue, a proliferation-inducing ligand (APRIL), modulate B cell functions by binding to three receptors, BAFFR, TACI and BCMA. APRIL binds more strongly to BCMA than to TACI while the opposite is true for BAFF.  In addition, BAFF, but not APRIL, binds to BAFF-R with high affinity.  Both BAFF and APRIL are known to form homotrimers and heterotrimers, which display different affinities for their cognate receptors.  To understand the structural basis underlying the different binding affinities, we solved the crystal structure of the APRIL-BAFF-BAFF heterotrimer, either alone or bound to TACI.  Analysis of these structures and comparative analysis of previously solved structures of BAFF and APRIL in complex with BAFFR, TACI or BCMA revealed that the charged residues of BAFF and APRIL are crucial determinants of receptor binding affinities, conferring homotrimers and heterotrimers with a variety of signaling strength to modulate responses of immune cells.

Protein folding in the cell is a complex process with a fine balance between productive and non-productive folding. To modulate, either up-regulating or down-regulating, the level of one specific protein with multiple approaches is possible, including the modulation of catalysed protein folding, the use of chemical and pharmacological chaperones, alteration of natural protein-protein interactions, the regulation of degradative pathways and manipulation of natural control mechanisms, such as the heat shock response and the unfolded protein response. Errors in proteostasis are linked to a wide range of disease states and many examples exist of the successful manipulation of proteostasis for the partial or complete elimination of the disease phenotype, including for many amyloid based diseases such as Parkinsons and Alzheimers as as well as for loss-of-function diseases such as Fabrys and Gauchers diseases. This review takes an overview of the different approaches that can be used to alter proteostasis with an emphasis on peptidomimetic inhibitors and activators of protein folding. It covers the modulators available, their mechanisms of action and potential limitations, including the problems of specificity in altering proteostasis.

DegS peptidase acts as a chaperone at low temperatures but switches to a peptidase (heat shock protein) at higher temperatures. Molecular chaperones and peptidases control the folded state of proteins by recognizing hydrophobic stretches of polypeptide that become exposed by misfolding or unfolding. They then bind these hydrophobic substrates to prevent aggregation or assist in protein refolding. If attempts at refolding fail, then irreversibly damaged proteins are degraded by peptidases such as DegS.

Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurode-generative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aβ42 peptide, in correlation with Nrf2 activation. Comparatively , lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aβ42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aβ oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.

Autophagy is a major cellular recycling process that delivers cellular material and entire organelles to lysosomes for degradation, in a selective or non-selective manner. This process is essential for the maintenance of cellular energy levels, components, and metabolites, as well as the elimination of cellular molecular damage, thereby playing an important role in numerous cellular activities. An important function of autophagy is to enable survival under starvation conditions and other stresses. The majority of factors implicated in aging are modifiable through the process of autophagy, including the accumulation of oxidative damage and loss of proteostasis, genomic instability and epigenetic alteration. These primary causes of damage could lead to mitochondrial dysfunction, deregulation of nutrient sensing pathways and cellular senescence, finally causing a variety of aging phenotypes. Remarkably, advances in the biology of aging have revealed that aging is a malleable process: a mild decrease in signaling through nutrient-sensing pathways can improve health and extend lifespan in all model organisms tested. Consequently, autophagy is implicated in both aging and age-related disease. Enhancement of the autophagy process is a common characteristic of all principal, evolutionary conserved anti-aging interventions, including dietary restriction, as well as inhibition of target of rapamycin (TOR) and insulin/IGF-1 signaling (IIS). As an emerging and critical process in aging, this review will highlight how autophagy can be modulated for health improvement.

Aging is one of the chief biomedical problems of the 21st century. After decades of basic research on biogerontology
(the science of aging), the aging process still remains an enigma. Although hundreds of "theories" on aging have
been formulated and many fundamental insights about age-related changes and genetic as well as environmental
interventions that change the pace of aging have been discovered, the actual why and how we age remain enigmatic.
In the post-genomic era there is an exponential increase in data. As a consequence it is a challenge to utilize all
information based on it and derive meaningful knowledge about biological phenomena. No individual scientist, no
group, nor consortium is capable of keeping up within their own field and are overwhelmed by the explosion of data
increase. Machine learning applied on biological data has the potential to solve this and cause a paradigm shift from
hypothesis-driven research (which predominates biological research including biogerontology) to data-driven
research.
This dissertation addresses this problem. In particular it proposes and executes the use of machine learning on
current existing data to predict drivers of aging (and therefore helps to distinguish causes from consequences),
interventions to counteract aging, and specific hypotheses to fill in research gaps that require experimental
validation.
The objective of this project is therefore to build computational models that are based on data relevant to the
phenomenon of aging and to predict as many of its aspects and dimensions as possible (thus elucidate their relations
to each other). For converting between and sorting within dimensions which are relevant to aging, different machine
learning models are evaluated. Ones models are built, it can be determined how much they can explain different
aspects of aging. Those models will also be capable of specifying which features are most relevant for prediction (in
both classification or regression). It is possible to train models that incorporate age-related changes based on
transcriptomic, proteomic, metabolomic, epigenomic as well as morphological data and their combinations. Machine
learning is further used to convert between and within them.
This work focuses on three types of predictors. Subsequently, discoveries are made with the statistical and learning
algorithms. The first model (lifespan predictor) is trained on predicting the lifespan based on genotype, environment
and combinations thereof. It is useful for predicting lifespan extending interventions on the population level. The
second model (age predictor) is trained on predicting the age given features measured on individuals. This is useful
for identifying biomarkers of aging and to determine the effects of interventions on the level of individuals. The third
model predicts functions/regulations of biological entities in regard to the aging process based on heterogeneous data
such as ontologies and diverse omics including time-series gene expression profiles (which can be visualized as
plots), and linked data. It is used to understand the role of genes and proteins as well as perhaps other entities such as
small molecules including lipids and other metabolites. Functions of proteins, which are still unknown, especially
those involved in yeast lipid metabolism and its regulation, can be predicted.
For this purpose we use primarily yeast as model organism as well as data on humans. Other biomedical model
organisms might be added if found beneficial.
The novel aspects of this research are for instance that 1) aging is investigated systematically in an unbiased
data-driven approach, 2) lifespan is predicted as continuous values, 3) age is predicted by combining multiple omics
data, 4) functions and regulations of biological entities like genes are predicted with high confidence from
heterogeneous data sources.
This thesis discovered that genetics is the most important feature of lifespan determination. Phenotypic features
related to lipid and membranes such as vacuolar morphology and autophagy activity are important for lifespan
determination according to the best performing models. A age predictor based on transcriptomics and proteomics can
highly accurately determine the age. It selected features are associated with both translation and lipid metabolism.
Among the top selected features are transcripts of genes when deleted exhibit abnormal vacuolar morphology as well
as targets of Opi1. Opi1 itself and its regulators were found to be differentially regulated post-transcriptional or
post-translational. Lastly, a function predictor for genes was created that achieved exceptional accuracy of
classifying aging genes. It learned for instance that piecemeal autophagy of the nucleus is strongly predictive for
aging-suppressor genes while cytoplasmic translation is strongly predictive for gerontogenes.
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Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 local-ization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cyto-plasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention , potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HN-RNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.

Unlike most checkpoint proteins, Mec1, an ATM/ATR kinase, is essential. We utilized mec1-4, a missense allele (E2130K) that confers diminished kinase activity, to interrogate the question. Unbiased screen for genetic interactors of mec1-4 identified numerous genes involved in proteostasis. mec1-4 confers sensitivity to heat, an amino acid analog, and Htt103Q, an aggregation-prone model peptide of Huntingtin. Oppositely, mec1-4 confers resistance to cycloheximide, a translation inhibitor. In response to heat, mec1-4 leads to widespread protein aggregation and cell death. Key components of the Mec1 signaling network, Rad53, Dun1, and Sml1, also impact survival in response to proteotoxic stress. Activation of autophagy or sml1Δ promotes aggregate resolution and rescues mec1-4 lethality. These findings show that proteostasis is a fundamental function of Mec1 and that Mec1 is likely to utilize its checkpoint response network to mediate resistance to proteotoxic stress, a role that may be...

Abstract: Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster...

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host–pathogen interactions that shape viral evol...

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichia coli and Acinetobacter baumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show th...

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show th...

The pathogenic drivers of sporadic and familial motor neuron disease (MND), such ALS, are unknown. MND impair the Ran GTPase cycle, which controls nucleocytoplasmic transport, ribostasis and proteostasis; however, cause-effect mechanisms of Ran GTPase modulators in motoneuron pathobiology are heretofore elusive. The cytosolic and peripheral nucleoporin, Ranbp2, is a critical regulator of the Ran GTPase cycle and proteostasis of neurological disease-prone substrates, but the roles of Ranbp2 in motoneuron biology and disease remain unknown. This study shows that conditional ablation of Ranbp2 in mouse Thy1-motoneurons causes ALS syndromes with hypoactivity followed by hind limb paralysis, respiratory distress and ultimately, death. These phenotypes are accompanied by declines of nerve conduction velocity, free fatty acids and phophatidylcholine of the sciatic nerve, g-ratios of sciatic and phrenic nerves, and hypertrophy of motoneurons. Further, Ranbp2 loss disrupts the nucleocytoplas...

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments.To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations.Reorganisation of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumu...

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidat...

Chaperones are protein complexes that help to fold and disaggregate a cell's proteins. It is not understood how four major chaperone systems of Escherichia coli work together in proteostasis: the recognition, sorting, folding, and disaggregating of the cell's many different proteins. Here, we model this machine. We combine extensive data on chaperoning, folding, and aggregation rates with expression levels of proteins and chaperones measured at different growth rates. We find that the proteostasis machine recognizes and sorts a client protein based on two biophysical properties of the client's misfolded state (M state): its stability and its kinetic accessibility from its unfolded state (U state). The machine is energy-efficient (the sickest proteins use the most ATP-expensive chaperones), comprehensive (it can handle any type of protein), and economical (the chaperone concentrations are just high enough to keep the whole proteome folded and disaggregated but no higher)....