ADAPs intrinsically disordered region is an actin sponge regulating T cell motility

Intrinsically disordered proteins (IDPs) play a vital role in biological processes that rely on transient molecular compartmentation1. In T cells, the dynamic switching between migration and adhesion mandates a high degree of plasticity in the interplay of adhesion and signaling molecules with the actin cytoskeleton2,3. Here, we show that the N-terminal intrinsically disordered region (IDR) of adhesion- and degranulation-promoting adapter protein (ADAP) acts as a multipronged scaffold for G- and F-actin, thereby promoting actin polymerization and bundling. Positively charged motifs, along a sequence of at least 200 amino acids, interact with both longitudinal sides of G-actin in a promiscuous manner. These polymorphic interactions with ADAP become constrained to one side once F-actin is formed. Actin polymerization by ADAP acts in synergy with a capping protein but competes with cofilin. In T cells, ablation of ADAP impairs adhesion and migration with a time-dependent reduction of the F-actin content in response to chemokine or T cell receptor (TCR) engagement. Our data suggest that IDR-assisted molecular crowding of actin above the critical concentration defines a new mechanism to regulate cytoskeletal dynamics. The principle of IDRs serving as molecular sponges to facilitate regulated self-assembly of filament-forming proteins might be a general phenomenon.

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

Liquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.

A C-TERMINAL ATAXIN-2 DISORDERED REGION PROMOTES HUNTINGTIN PROTEIN AGGREGATION AND NEURODEGENERATION IN DROSOPHILA MODELS OF HUNTINGTON'S DISEASE

The Ataxin-2 (Atx2) protein contributes to the progression of neurodegenerative phenotypes in animal models of amyotrophic lateral sclerosis (ALS), type 2 spinocerebellar ataxia (SCA-2), Parkinson’s Disease (PD) and Huntington’s Disease (HD). However, because the Atx2 protein contains multiple separable activities, deeper understanding requires experiments to address the exact mechanisms by which Atx2 modulates neurodegeneration (ND) progression. Recent work on two ALS models, C9ORF72 and FUS, in Drosophila has shown that a C-terminal intrinsically disordered region (cIDR) of Atx2 protein, required for assembly of ribonucleoprotein (RNP) granules, is essential for the progression of neurodegenerative phenotypes as well as for accumulation of protein inclusions associated with these ALS models. Here we show that the Atx2-cIDR also similarly contributes to the progression of degenerative phenotypes and accumulation of Huntingtin protein aggregates in Drosophila models of HD. Because Huntingtin is not an established component of RNP granules, these observations support a recently hypothesised, unexpected protein-handling function for RNP granules, which could contribute to the progression of Huntington’s disease and, potentially, other proteinopathies.

Ataxin-2 Disordered Region Promotes Huntingtin Protein Aggregation And Neurodegeneration In Drosophila Models Of Huntington’s Disease

ABSTRACTThe Ataxin-2 (Atx2) protein contributes to the progression of neurodegenerative phenotypes in animal models of amyotrophic lateral sclerosis (ALS), type 2 spinocerebellar ataxia (SCA-2), Parkinson’s Disease (PD) and Huntington’s Disease (HD). However, because the Atx2 protein contains multiple separable activities, deeper understanding requires experiments to address the exact mechanisms by which Atx2 modulates neurodegeneration (ND) progression. Recent work on two ALS models, C9ORF72 and FUS, in Drosophila has shown that a C-terminal intrinsically disordered region (cIDR) of Atx2 protein, required for assembly of ribonucleoprotein (RNP) granules, is essential for the progression of neurodegenerative phenotypes as well as for accumulation of protein inclusions associated with these ALS models. Here we show that the Atx2-cIDR also similarly contributes to the progression of degenerative phenotypes and accumulation of Huntingtin protein aggregates in Drosophila models of HD. Because Huntingtin is not an established component of RNP granules, these observations support a recently hypothesised, unexpected protein-handling function for RNP granules, which could contribute to the progression of Huntington’s disease and, potentially, other proteinopathies.

The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability

The mammalian CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that has emerged as an important player in protecting genome stability and preserving telomere integrity. Studies have shown that CST localizes at stalled replication forks and is critical for protecting the stability of nascent strand DNA. Recent cryo-EM analysis reveals that CST subunits possess multiple OB-fold domains that can form a decameric supercomplex. While considered to be RPA-like, CST acts distinctly from RPA to protect genome stability. Here, we report that while the OB domain of STN1 shares structural similarity with the OB domain of RPA32, the STN1-OB domain contains an intrinsically disordered region (IDR) that is important for maintaining genome stability under replication stress. Single mutations in multiple positions in this IDR, including cancer-associated mutations, cause genome instabilities that are elevated by replication stress and display reduced cellular viability and increased HU sensitivity. While IDR mutations do not impact CST complex formation or CST interaction with its binding partner RAD51, they diminish RAD51 foci formation when replication is perturbed. Interestingly, the IDR is critical for STN1–POLα interaction. Collectively, our results identify the STN1 IDR as an important element in regulating CST function in genome stability maintenance.

Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid–liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.