Hallmarks of cotranslational protein complex assembly and its relationship with the dominant-negative effect

Proteins carry out most of the biochemical phenomena necessary for life as we know it. The majority of proteins do not function alone in the cell, but are instead subunits that assemble into complexes with copies of themselves and other proteins. For decades, due to limited evidence to support otherwise, the textbook model was that subunits have to be fully synthesised before they diffuse away and collide randomly with their partners to from a complex. More recently, however, increasing evidence has accumulated, revealing that this model is incomplete. We now understand that many subunits begin the assembly process during their translation on the ribosome. This phenomenon has important implications for the structure, function, and evolution of protein complexes, as well as for the understanding and the prediction of the mechanisms by which genetic mutations cause disease. The first chapter provides an overview of our current understanding of how and why proteins assemble into complexes. Two classes of complexes are discussed: homomers, which consist of genetically identical copies of a protein and exhibit structural symmetry, and heteromers, which involve the assembly of non-identical proteins and are more common in human cells. I review the historical experiments that contributed to the discovery of cotranslational assembly, including recent breakthroughs that have made its proteome-wide detection possible, which is of tremendous value to this thesis. I provide an overview of genetic mutations in the context of human disease, as the present work has considerable clinical applications beyond its contribution to fundamental biology. In the second chapter, I investigate the properties of subunit interfaces that influence cotranslational assembly using a combination of proteomic, structural, and computational approaches. I show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, I compare the sizes of first and last translated interfaces of heteromeric subunits in the proteomes of three evolutionary distant species. This analysis reveals that N-terminal interfaces, on average, tend to be larger than C-terminal interfaces. Notably, the trend is significant in ancient subunits or those organised into operons in bacteria, suggesting that large N-terminal interfaces may have been selected for to seed the assembly pathway cotranslationally. The third chapter explores an important hypothesis regarding cotranslational assembly: can it counter the dominant-negative effect, whereby the co-assembly of mutant and wild-type subunits impairs the activity of a protein complex? First, I show that cotranslationally assembling subunits are much less likely to be associated with autosomal dominant relative to recessive disorders. Second, I observe that subunits with dominant-negative disease mutations are significantly depleted in cotranslational assembly compared to those associated with loss-of-function mutations. Additionally, I find that complexes with known dominant-negative effects tend to expose their interfaces later during translation, lessening the likelihood of cotranslational assembly. Altogether, I find strong support for the hypothesis that the allele-specific nature of cotranslational assembly can buffer the effect of certain dominant mutations. In the fourth chapter, I synthesize the hallmarks of cotranslational assembly and discuss their mechanistic interpretations, highlighting the differences between neutralist and selectionist perspectives regarding their functional importance. Finally, in the fifth chapter, by combining a diverse range of gene-level features, I train a computational model for predicting proteins likely to be associated with non-loss-of-function (non-LOF) disease mechanisms, with the aim of accelerating the discovery of novel disease variants. I first generate a model that utilizes protein complex structural data and showcase its ability to detect properties explicitly absent from the model but are linked to proteins that give rise to non-LOF disease mechanisms. Although the results reflect the idea that LOF and non-LOF mechanisms can be captured at the protein-level, the predictor is strongly limited by the availability of protein complex structural data. Due to this limitation, I introduce a new model architecture with a spectrum of surrogate features, notably excluding those based on experimental protein complex structure data. The resulting models enable the estimation of probabilities for a protein exhibiting loss-of-function, gain-of-function, and dominant-negative molecular disease mechanisms across the entire proteome. In preliminary results, I demonstrate the practical applications of these models, including the prioritization of mutations with non-LOF-like properties in population genetic data and the detection of cryptic de novo dominant-negative mutations in developmental disorders. This thesis offers fresh insights into the molecular and evolutionary aspects of cotranslational assembly and its role in human disease

Large protein complex interfaces have evolved to promote cotranslational assembly

Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromeric subunits in bacterial, yeast, and human complexes. When considering all together, we observe the N-terminal interface to be larger than the C-terminal interface 54% of the time, increasing to 64% when we exclude subunits with only small interfaces, which are unlikely to cotranslationally assemble. This strongly suggests that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding

Type I interferonopathy due to a homozygous loss-of-inhibitory-function mutation in STAT2

International audiencePurpose STAT2 is both an effector and negative regulator of type I interferon (IFN-I) signalling. We describe the characterization of a novel homozygous missense STAT2 substitution in a patient with a type I interferonopathy. Methods Whole-genome sequencing (WGS) was used to identify the genetic basis of disease in a patient with features of enhanced IFN-I signalling. After stable lentiviral reconstitution of STAT2-null human fibrosarcoma U6A cells with STAT2 wild type or p.(A219V), we performed quantitative polymerase chain reaction, western blotting, immunofluorescence, and co-immunoprecipitation to functionally characterize the p.(A219V) variant. Results WGS identified a rare homozygous single nucleotide transition in STAT2 (c.656C > T), resulting in a p.(A219V) substitution, in a patient displaying developmental delay, intracranial calcification, and up-regulation of interferon-stimulated gene (ISG) expression in blood. In vitro studies revealed that the STAT2 p.(A219V) variant retained the ability to transduce an IFN-I stimulus. Notably, STAT2 p.(A219V) failed to support receptor desensitization, resulting in sustained STAT2 phosphorylation and ISG up-regulation. Mechanistically, STAT2 p.(A219V) showed defective binding to ubiquitin specific protease 18 (USP18), providing a possible explanation for the chronic IFN-I pathway activation seen in the patient. Conclusion Our data indicate an impaired negative regulatory role of STAT2 p.(A219V) in IFN-I signalling and that mutations in STAT2 resulting in a type I interferonopathy state are not limited to the previously reported R148 residue. Indeed, structural modelling highlights at least 3 further residues critical to mediating a STAT2-USP18 interaction, in which mutations might be expected to result in defective negative feedback regulation of IFN-I signalling

Interface Gain-of-Function Mutations in TLR7 Cause Systemic and Neuro-inflammatory Disease

TLR7 recognizes pathogen-derived single-stranded RNA (ssRNA), a function integral to the innate immune response to viral infection. Notably, TLR7 can also recognize self-derived ssRNA, with gain-of-function mutations in human TLR7 recently identified to cause both early-onset systemic lupus erythematosus (SLE) and neuromyelitis optica. Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I substitution arose de novo, the F507S mutation was present in three individuals from the same family, including a severely affected male, notably given that the TLR7 gene is situated on the X chromosome and that all other cases so far described have been female. The observation of mutations at residues 507 and 528 of TLR7 indicates the importance of the TLR7 dimerization interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. Finally, while mutations in TLR7 can result in SLE-like disease, our data suggest a broader phenotypic spectrum associated with TLR7 gain-of-function, including significant neurological involvement

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence

Summary. Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2− breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients

Large protein complex interfaces have evolved to promote cotranslational assembly

Statistical analysis in R languag

Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases

Data and analysis cod

Expanding the neurodevelopmental phenotype associated with HK1 de novo heterozygous missense variants

Neurodevelopmental disorder with visual defects and brain anomalies (NEDVIBA) is a recently described genetic condition caused by de novo missense HK1 variants. Phenotypic data is currently limited; only seven patients have been published to date. This descriptive case series of a further four patients with de novo missense HK1 variants, alongside integration of phenotypic data with the reported cases, aims to improve our understanding of the associated phenotype. We provide further evidence that de novo HK1 variants located within the regulatory-terminal domain and alpha helix are associated with neurological problems and visual problems. We highlight for the first time an association with a raised cerebrospinal fluid lactate and specific abnormalities to the basal ganglia on brain magnetic resonance imaging, as well as associated respiratory issues and swallowing/feeding difficulties. We propose that this distinctive neurodevelopmental phenotype could arise through disruption of the regulatory glucose-6-phosphate binding site and subsequent gain of function of HK1 within the brain