Polyserine repeats promote coiled coil-mediated fibril formation and length-dependent protein aggregation.

Short polyserine (polyS) repeats are frequently found in proteins and longer ones are produced in neurological disorders such as Huntington disease (HD) owing to translational frameshifting or non-ATG-dependent translation, together with polyglutamine (polyQ) and polyalanine (polyA) repeats, forming intracellular aggregates. However, the physiological and pathological structures of polyS repeats are not clearly understood. Early studies highlighted their structural versatility, similar to other homopolymers whose conformation is influenced by the surrounding protein context. As polyS stretches are frequently near polyQ and polyA repeats, which can be part of coiled coil (CC) structures, and the frameshift-derived polyS repeats in HD directly flank CC heptads important for aggregation, we investigate here the structural and aggregation properties of polyS in the context of CC structures. We have taken advantage of peptide models, previously used to study polyQ and polyA in CCs, in which we inserted polyS repeats of variable length and studied them in comparison with polyQ and polyA peptides. We found that polyS repeats promote CC-mediated polymerization and fibrillization as revealed by circular dichroism, chemical crosslinking, and atomic force microscopy. Furthermore, they promote CC-based, length-dependent intracellular aggregation, which is negligible with 7 and widespread with 49 serines. These findings show that polyS repeats can participate in the formation of CCs, as previously found for polyQ and polyA, conferring to peptides distinctive structural properties with aggregation kinetics that are intermediate between those of polyA and polyQ CCs, and contribute to an overall structural definition of the pathophysiogical roles of homopolymeric repeats in CC structures.

Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.

The expansion of homopolymeric glutamine (polyQ) or alanine (polyA) repeats in certain proteins owing to genetic mutations induces protein aggregation and toxicity, causing at least 18 human diseases. PolyQ and polyA repeats can also associate in the same proteins, but the general extent of their association in proteomes is unknown. Furthermore, the structural mechanisms by which their expansion causes disease are not well understood, and these repeats are generally thought to misfold upon expansion into aggregation-prone ß-sheet structures like amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) structures in triggering aggregation and toxicity of polyQ-expanded proteins, raising the possibility that polyA repeats may as well form these structures, by themselves or in association with polyQ. We found through bioinformatics screenings that polyA, polyQ and polyQA repeats have a phylogenetically graded association in human and non-human proteomes and associate/overlap with CC domains. Circular dichroism and cross-linking experiments revealed that polyA repeats can form--alone or with polyQ and polyQA--CC structures that increase in stability with polyA length, forming higher-order multimers and polymers in vitro. Using structure-guided mutagenesis, we studied the relevance of polyA CCs to the in vivo aggregation and toxicity of RUNX2--a polyQ/polyA protein associated with cleidocranial dysplasia upon polyA expansion--and found that the stability of its polyQ/polyA CC controls its aggregation, localization and toxicity. These findings indicate that, like polyQ, polyA repeats form CC structures that can trigger protein aggregation and toxicity upon expansion in human genetic diseases.

PolyQ length-based molecular encoding of vocalization frequency in FOXP2.

The transcription factor FOXP2, a regulator of vocalization- and speech/language-related phenotypes, contains two long polyQ repeats (Q1 and Q2) displaying marked, still enigmatic length variation across mammals. We found that the Q1/Q2 length ratio quantitatively encodes vocalization frequency ranges, from the infrasonic to the ultrasonic, displaying striking convergent evolution patterns. Thus, species emitting ultrasonic vocalizations converge with bats in having a low ratio, whereas species vocalizing in the low-frequency/infrasonic range converge with elephants and whales, which have higher ratios. Similar, taxon-specific patterns were observed for the FOXP2-related protein FOXP1. At the molecular level, we observed that the FOXP2 polyQ tracts form coiled coils, assembling into condensates and fibrils, and drive liquid-liquid phase separation (LLPS). By integrating evolutionary and molecular analyses, we found that polyQ length variation related to vocalization frequency impacts FOXP2 structure, LLPS, and transcriptional activity, thus defining a novel form of polyQ length-based molecular encoding of vocalization frequency.

Compound Dynamics and Combinatorial Patterns of Amino Acid Repeats Encode a System of Evolutionary and Developmental Markers.

Homopolymeric amino acid repeats (AARs) like polyalanine (polyA) and polyglutamine (polyQ) in some developmental proteins (DPs) regulate certain aspects of organismal morphology and behavior, suggesting an evolutionary role for AARs as developmental "tuning knobs." It is still unclear, however, whether these are occasional protein-specific phenomena or hints at the existence of a whole AAR-based regulatory system in DPs. Using novel approaches to trace their functional and evolutionary history, we find quantitative evidence supporting a generalized, combinatorial role of AARs in developmental processes with evolutionary implications. We observe nonrandom AAR distributions and combinations in HOX and other DPs, as well as in their interactomes, defining elements of a proteome-wide combinatorial functional code whereby different AARs and their combinations appear preferentially in proteins involved in the development of specific organs/systems. Such functional associations can be either static or display detectable evolutionary dynamics. These findings suggest that progressive changes in AAR occurrence/combination, by altering embryonic development, may have contributed to taxonomic divergence, leaving detectable traces in the evolutionary history of proteomes. Consistent with this hypothesis, we find that the evolutionary trajectories of the 20 AARs in eukaryotic proteomes are highly interrelated and their individual or compound dynamics can sharply mark taxonomic boundaries, or display clock-like trends, carrying overall a strong phylogenetic signal. These findings provide quantitative evidence and an interpretive framework outlining a combinatorial system of AARs whose compound dynamics mark at the same time DP functions and evolutionary transitions.

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping the interaction networks of the human proteome, and define proteome-wide knowledge that may guide the informed biological exploration of the role of AARs in protein interactions.

Synaptic vesicles are "primed" for fast clathrin-mediated endocytosis at the ribbon synapse.

Retrieval of synaptic vesicles can occur 1-10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone. The subsequent loss of clathrin from these regions reflected endocytosis because the application of a potent clathrin inhibitor Pitstop2 dramatically slowed down this phase by ~3 fold. These results indicate that clathrin-dependent retrieval of synaptic vesicles is unusually fast, most probably because of a "priming" step involving a state of association of clathrin with the docked vesicle and with the endosomes and cisternae surrounding the ribbons. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) showed that the majority of clathrin is moving with the same kinetics as synaptic vesicle proteins. Together, these results indicate that the fast endocytic mechanism operating to retrieve synaptic vesicles differs substantially from the classical mode of CME operating via formation of a coated pit.

Synaptic ribbons: machines for priming vesicle release?

Synaptic ribbons are specialized organelles that hold vesicles close to the active zone of sensory synapses, but their function is mysterious. Acute disruption of the ribbon complex using light has now revealed that it has a role in priming synaptic vesicles for fusion.