Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.© 2023. The Author(s)

Genetische und chemische hydrophobe Modifikation von Transkriptionsfaktor-Kondensaten bei menschlichen Erkrankungen

The work in this thesis examines how hydrophobic modifications within intrinsically disordered regions (IDRs) of transcription factors (TFs), caused by genetic mutations and drug-induced changes, disrupt TF condensation and gene expression programs in various human disease states. I establish as a general paradigm that disease-associated poly-alanine repeat expansions promote the formation of homotypic TF condensates with gel-like material properties, at the expense of heterotypic interactions with other components of the transcriptional apparatus. This effect was elicited by hydrophobic repeat expansions found in the following developmental disorders: synpolydactyly (SPD), cleidocranial dysplasia (CCD), and hand-foot-genital-syndrome (HFGS). Utilizing insights from this paradigm, I hypothesized that the condensation capacity of TFs may be controlled with chemical modifiers of TF-IDRs, in efforts to treat diseases reliant on aberrant transcriptional programs, such as cancer. To this end, this thesis provides evidence that the condensation of an oncogenic transcription factor, the androgen receptor (AR), can be targeted with small molecules that selectively partition into condensates formed by the disordered activation domain of the AR. Increasing the hydrophobicity of the small molecule resulted in higher potency in the arrest of proliferation and AR-driven gene expression programs in a human model of prostate cancer (PCa). Together, these results suggest that hydrophobic modification of transcription factors and small molecules that partition into condensates can be leveraged for therapeutic intent.In dieser Dissertation wurde untersucht, wie hydrophobe Modifikationen innerhalb von intrinsisch ungeordneten Regionen (IDRs) von Transkriptionsfaktoren (TFs), verursacht durch genetische Mutationen und arzneimittelinduzierte Veränderungen, die TF- Kondensation und Genexpressionsprogramme in verschiedenen menschlichen Krankheitsstadien stören. Dadurch konnte etabliert werden, dass krankheitsassoziierte Poly- Alanin-Expansionen die Bildung von homotypischen TF-Kondensaten mit gelartigen Materialeigenschaften fördern, auf Kosten heterotypischer Wechselwirkungen mit anderen Komponenten des Transkriptionsapparates. Dieser Effekt wurde in folgenden Entwicklungsstörungen charakterisiert: Synpolydaktylie (SPD), cleidocraniale Dysplasie (CCD) und Hand-Fuß-Genital-Syndrom (HFGS). Basierend auf diesen Erkenntnissen folgte die Hypothese, dass die Kondensationskapazität von TFs mit chemischen Modifikationen von TF-IDRs kontrolliert werden kann, um Krankheiten wie z.B. Krebs zu behandeln, die auf abweichende Transkriptionsprogramme angewiesen sind. In diesem Zusammenhang konnte ich in meiner Dissertation belegen, dass die Kondensation eines onkogenen Transkriptionsfaktors, des Androgenrezeptors (AR), mit kleinen Molekülen moduliert werden kann, die sich selektiv in Kondensate integrieren, die von der ungeordneten Aktivierungsdomäne des AR gebildet werden. Eine Erhöhung der Hydrophobie dieses Moleküls führte zu einer stärkeren Hemmung von Proliferations- und AR-gesteuerten Genexpressionsprogrammen, welche in einem menschlichen Prostatakrebsmodell (PCa) nachgewiesen wurde. Zusammengenommen legen diese Ergebnisse nahe, dass die hydrophobe Modifikation von Transkriptionsfaktoren und kleine Moleküle, die sich in Kondensate integrieren, für therapeutische Zwecke genutzt werden kann

Self-oligomerization of the hydrophobic regions within amyloid beta and prion protein

Proteinopathies of the central nervous system (CNS) encompass a broad range of disorders (Alzheimer, Parkinson, Huntington and prion diseases) which invariably lead to human neurodegeneration. Such diseases bear a heavy financial burden on healthcare and elevate socio-economic costs, and will continue to do so as the general population ages. In our current predicament, there is a lack in etiological treatments for such diseases, rendering them incurable and unpreventable. Consensus in the field of translational research has attributed disease pathology to the misfolding and aggregation of certain proteins. The work in this thesis examines two highly aggregating proteins found in Alzheimer and Prion diseases, beta amyloid (Aβ) and prion protein (PrP) respectively, in efforts to assist in the development of rationally designed therapies. Intriguingly, both proteins possess highly hydrophobic stretches rich in small amino acids such as glycine (G) and alanine (A), which can organize to form motifs that determine their synthesis, trafficking, folding, processing and degradation in cells. The guiding hypothesis is that such regions in these proteins may provide for a valid molecular target for therapeutic intervention.Recently, a human mutation within the highly conserved hydrophobic region of PrP (glycine 127 exchanged to valine) was characterized to confer complete neuroprotection from infectious prions. Similarly, other familial mutations and polymorphisms which exist in this very region have been shown to elicit or potentiate various prion disease phenotypes. Interestingly, of the mutations cataloged to date, the pathogenic and protective mutations seem to exist separately at two distinct motifs within the hydrophobic region of PrP: namely its N-terminal AGAAAAGA palindrome and C-terminal GxxxG-like motifs. In efforts to understand how such mutations alter the behavior of PrP, I examined their effects on oligomerization of the hydrophobic region within the lipid bilayer environment. As the entirety of the region is heavily implicated in the biogenesis and misfolding of PrP, the findings described here may help provide mechanistic insight on how one could develop a therapeutic agent that could target these motifs to exert effects much like the G127V mutation. Like PrP, Aβ also possesses a hydrophobic region notorious for aggregation and involvement in neurodegeneration. This region also contains a triple repeat GxxxG motif, which has been dually implicated in the biogenesis and toxicity of Aβ peptides. As such, I have examined certain compounds that can modulate generation of these peptides (γ-secretase modulators), to examine if they can interact with GxxxG motifs to elicit their therapeutic effects. In addition to this, I have also developed a facile method for the production of the most toxic and highly aggregating Aβ species, Aβ 1 - 42. As synthetically derived Aβ counterparts have lower toxicity, slower aggregation kinetics, and are commercially inflated in price, this production method for recombinant Aβ will help expedite our efforts in the realization of a molecular candidate that can neutralize the toxicity of this peptide; an integral strategy for the treatment of Alzheimer disease.Les protéinopathies du système nerveux central (SNC) englobent une large gamme de troubles (maladie d'Alzheimer, Parkinson, Huntington et maladie à prions) qui conduisent invariablement à une neurodégénérescence humaine. De telles maladies ont un lourd poids financier pour les soins de santé et augmentent les coûts socioéconomiques, et continueront à faire à mesure que la population générale vieillira. Dans notre situation actuelle, il y a un manque de traitements étiologiques pour de telles maladies, les rendant incurables et inévitables. Le consensus établi par le domaine de la recherche translationnelle attribue la pathologie au repli et à l'agrégation de certaines protéines. Ce travail de thèse examine deux protéines hautement agrégées trouvées dans les maladies d'Alzheimer et à prions, la protéine β-amyloïde (Aβ) et la protéine prion (PrP), dans le but d'aider au développement de thérapies rationnellement conçues. De façon intéressante, les deux protéines possèdent des domaines hautement hydrophobes riches en petits acides aminés tels que la glycine (G) et l'alanine (A), qui peuvent s'organiser pour former des motifs qui déterminent leur synthèse, leur trafic, leur repliement, leur transport et leur dégradation dans les cellules. L'hypothèse directrice est que de ces domaines pourraient fournir une cible moléculaire valide sur le plan thérapeutique.Récemment, une mutation humaine dans la région hydrophobe hautement conservée de PrP (glycine 127 remplacée par une valine) a été caractérisée. Elle entraine une neuroprotection complète contre les infections par les prions. De même, d'autres mutations familiales et des polymorphismes qui existent dans cette même région ont été montrés pour entrainer ou potentialiser divers phénotypes de la maladie à prions. Parmi les mutations mises en évidence à ce jour, il est intéressant d'bbserver que les mutations pathogènes et protectrices semblent exister séparément dans deux motifs distincts dans la région hydrophobe de PrP, à savoir : le palindrome N-terminal AGAAAAGA et les motifs C-terminaux GxxxG. Dans le but de comprendre comment de telles mutations altèrent le comportement de la PrP, j'ai examiné leurs effets sur l'oligomérisation de la région hydrophobe dans l'environnement de la bicouche lipidique. Étant donné que la totalité de la région est fortement impliquée dans la biogenèse et le repliement de la PrP, les découvertes décrites ici peuvent aider à fournir un aperçu mécanistique de la façon dont on pourrait développer un agent thérapeutique qui pourrait cibler ces motifs afin d'exercer des effets similaires à ceux de la mutation G127V.Comme PrP, Aβ possède également une région hydrophobe connue pour avoir un rôle dans l'agrégation et être impliquée dans la neurodégénérescence. Cette région contient également un motif GxxxG à répétition triple, impliqué dans la biogenèse et la toxicité des peptides Aß. Ainsi, j'ai examiné certains composés qui peuvent moduler la génération de ces peptides (modulateurs des γ-sécrétases), afin de determiner s'ils sont capables d'interagir avec des motifs GxxxG et induire leurs effets thérapeutiques. De plus, j'ai développé une méthode simple de production des espèces Aβ les plus toxiques et possédant une capacité élevée à s'agréger, Aβ 1 - 42. Comme les dérivés homologues de synthèse de Aβ ont une toxicité plus faible, une cinétique d'agrégation plus lente et des prix très élevés sur le marché, cette méthode de production pour l'Aß recombinant aiderait à accélérer nos efforts dans l'élaboration d'un candidat moléculaire qui pourrait neutraliser la toxicité de ce peptide ; une stratégie complète pour le traitement de la maladie d'Alzheimer

Narrowband searches for continuous and long-duration transient gravitational waves from known pulsars in the LIGO-Virgo third observing run

Isolated neutron stars that are asymmetric with respect to their spin axis are possible sources of detectable continuous gravitational waves. This paper presents a fully coherent search for such signals from eighteen pulsars in data from LIGO and Virgo's third observing run (O3). For known pulsars, efficient and sensitive matched-filter searches can be carried out if one assumes the gravitational radiation is phase-locked to the electromagnetic emission. In the search presented here, we relax this assumption and allow both the frequency and the time derivative of the frequency of the gravitational waves to vary in a small range around those inferred from electromagnetic observations. We find no evidence for continuous gravitational waves, and set upper limits on the strain amplitude for each target. These limits are more constraining for seven of the targets than the spin-down limit defined by ascribing all rotational energy loss to gravitational radiation. In an additional search, we look in O3 data for long-duration (hours–months) transient gravitational waves in the aftermath of pulsar glitches for six targets with a total of nine glitches. We report two marginal outliers from this search, but find no clear evidence for such emission either. The resulting duration-dependent strain upper limits do not surpass indirect energy constraints for any of these targets

Narrowband Searches for Continuous and Long-duration Transient Gravitational Waves from Known Pulsars in the LIGO-Virgo Third Observing Run

Isolated neutron stars that are asymmetric with respect to their spin axis are possible sources of detectable continuous gravitational waves. This paper presents a fully coherent search for such signals from eighteen pulsars in data from LIGO and Virgo’s third observing run (O3). For known pulsars, efficient and sensitive matched-filter searches can be carried out if one assumes the gravitational radiation is phase-locked to the electromagnetic emission. In the search presented here, we relax this assumption and allow both the frequency and the time derivative of the frequency of the gravitational waves to vary in a small range around those inferred from electromagnetic observations. We find no evidence for continuous gravitational waves, and set upper limits on the strain amplitude for each target. These limits are more constraining for seven of the targets than the spin-down limit defined by ascribing all rotational energy loss to gravitational radiation. In an additional search, we look in O3 data for long-duration (hours–months) transient gravitational waves in the aftermath of pulsar glitches for six targets with a total of nine glitches. We report two marginal outliers from this search, but find no clear evidence for such emission either. The resulting duration-dependent strain upper limits do not surpass indirect energy constraints for any of these targets

Searches for Gravitational Waves from Known Pulsars at Two Harmonics in the Second and Third LIGO-Virgo Observing Runs

We present a targeted search for continuous gravitational waves (GWs) from 236 pulsars using data from the third observing run of LIGO and Virgo (O3) combined with data from the second observing run (O2). Searches were for emission from the l = m = 2 mass quadrupole mode with a frequency at only twice the pulsar rotation frequency (single harmonic) and the l = 2, m = 1, 2 modes with a frequency of both once and twice the rotation frequency (dual harmonic). No evidence of GWs was found, so we present 95% credible upper limits on the strain amplitudes h 0 for the single-harmonic search along with limits on the pulsars’ mass quadrupole moments Q 22 and ellipticities ε. Of the pulsars studied, 23 have strain amplitudes that are lower than the limits calculated from their electromagnetically measured spin-down rates. These pulsars include the millisecond pulsars J0437−4715 and J0711−6830, which have spin-down ratios of 0.87 and 0.57, respectively. For nine pulsars, their spin-down limits have been surpassed for the first time. For the Crab and Vela pulsars, our limits are factors of ∼100 and ∼20 more constraining than their spin-down limits, respectively. For the dual-harmonic searches, new limits are placed on the strain amplitudes C 21 and C 22. For 23 pulsars, we also present limits on the emission amplitude assuming dipole radiation as predicted by Brans-Dicke theory

Searches for gravitational waves from known pulsars at two harmonics in the second and third LIGO-Virgo observing runs

We present a targeted search for continuous gravitational waves (GWs) from 236 pulsars using data from the third observing run of Advanced LIGO and Virgo (O3) combined with data from the second observing run (O2). Searches were for emission from the l=m=2 mass quadrupole mode with a frequency at only twice the pulsar rotation frequency (single harmonic) and the l=2, m=1,2 modes with a frequency of both once and twice the rotation frequency (dual harmonic). No evidence of GWs was found so we present 95% credible upper limits on the strain amplitudes h_0 for the single harmonic search along with limits on the pulsars' mass quadrupole moments Q_{22} and ellipticities \varepsilon. Of the pulsars studied, 23 have strain amplitudes lower than the limits calculated from their electromagnetically measured spin-down rates. These pulsars include the millisecond pulsars J0437\textminus4715 and J0711\textminus6830 which have spin-down ratios of 0.87 and 0.57 respectively. For nine pulsars, their spin-down limits have been surpassed for the first time. For the Crab and Vela pulsars our limits are factors of \sim 100 and \sim 20 more constraining than their spin-down limits, respectively. For the dual harmonic searches, new limits are placed on the strain amplitudes C_{21} and C_{22}. For 23 pulsars we also present limits on the emission amplitude assuming dipole radiation as predicted by Brans-Dicke theory