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Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Ataxia cause identified An investigation into the molecular basis of the disease SCAN1 (spinocerebellar ataxia with axonal neuropathy-1) has identified for the first time a defect in the repair of chromosomal single-strand breaks in a neurodegenerative disease. The disease results from mutations in tyrosyl phosphodiesterase 1, but the known function of this enzyme — repairing double-strand breaks during replication — seemed unlikely to cause the observed pathology. The new study reveals a second function for the enzyme in human cells: repairing chromosome breaks caused by oxidative stress in post-mitotic neurons, and it is this that is likely to cause the symptoms of SCAN-1. Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1) 1 . In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells 2 , 3 , 4 . Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype 4 , 5 , 6 , 7 . Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8 ). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIα and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.

Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid–liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA–protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.Recent studies have highlighted the contribution of RNA to cellular liquid–liquid phase separation and condensate formation. RNA features modulate the composition and biophysical properties of RNA–protein condensates, which have various cellular functions, including RNA transport and localization, supporting catalytic processes and responding to stress.

Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.Biomolecular condensates are membraneless molecular assemblies formed via liquid–liquid phase separation. They have a plethora of roles, ranging from controlling biochemical reactions to regulating cell organization and cell function. This article provides a framework for the study of condensate functions across these cellular length scales, offering to bring new understanding of biological processes.

Genomic DNA is folded into loops and topologically associating domains (TADs), which serve important structural and regulatory roles. It has been proposed that these genomic structures are formed by a loop extrusion process, which is mediated by structural maintenance of chromosomes (SMC) protein complexes. Recent single-molecule studies have shown that the SMC complexes condensin and cohesin are indeed able to extrude DNA into loops. In this Review, we discuss how the loop extrusion hypothesis can explain key features of genome architecture; cellular functions of loop extrusion, such as separation of replicated DNA molecules, facilitation of enhancer–promoter interactions and immunoglobulin gene recombination; and what is known about the mechanism of loop extrusion and its regulation, for example, by chromatin boundaries that depend on the DNA binding protein CTCF. We also discuss how the loop extrusion hypothesis has led to a paradigm shift in our understanding of both genome architecture and the functions of SMC complexes.Chromatin loops are proposed to be formed through loop extrusion by structural maintenance of chromosomes (SMC) complexes. Recent studies have shown that the SMC complexes condensin and cohesin are indeed able to extrude DNA, and caused a paradigm shift in our understanding of genome organization and the cellular functions of SMC complexes.

Many biomolecules undergo liquid–liquid phase separation to form liquid-like condensates that mediate diverse cellular functions 1 , 2 . Autophagy is able to degrade such condensates using autophagosomes—double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast 3 – 5 . Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein–protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. The pre-autophagosomal structure in yeast is a liquid-like condensate of Atg proteins whose phase separation may have a critical, active role in autophagy.

Electron cryo-microscopy structures of the human peptide-loading complex shed light on its operation and on the onset of adaptive immune responses. Structure of a peptide loader The peptide-loading complex (PLC) is a dynamic membrane complex in the endoplasmic reticulum that regulates the transport and loading of antigenic peptides onto major histocompatibility complex class I (MHC-I) molecules. As such, this complex has a key role in important adaptive immune responses to infections and tumour progression. Here, Robert Tampé and colleagues report the structure of the human PLC by electron cryo-microscopy. The editing modules of the complex are centred around the TAP transporter, which delivers the peptides from the cytosol, and peptide loading appears to induce changes in the structure of MHC-I, releasing the stable peptide/MHC-I complexes from the PLC. This provides glimpses into the mechanism of the PLC, antigen processing and the onset of MHC-I-mediated immunity. The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide–MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells 1 , 2 . Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin 3 , 4 . The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt’s lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4 CRBN ) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4 CRBN . Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4 CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4 CRBN . Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4 CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins. The crystal structures of thalidomide and its derivatives bound to the E3 ligase subcomplex DDB1–CRBN are shown; these drugs are found to have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins. Thalidomide's dual mechanism of action Introduced in Europe in 1957 as a mild sedative, thalidomide was widely used in pregnant women as a treatment for morning sickness. This led to the birth of thousands of children with multiple defects and the drug was withdrawn in 1962. Since then thalidomide and its derivatives have emerged as effective treatments for the cancer multiple myeloma and the associated disorder 5q-dysplasia. The primary teratogenic target of thalidomide is cereblon (CRBN), part of E3 ubiquitin ligase complex CUL4–RBX1–DDB1–CRBN (CRL4 CRBN ). Here, Nicolas Thomä and colleagues present the crystal structure of DDB1–CRBN E3 ubiquitin ligase bound to thalidomide and to the related drugs lenalidomide and pomalidomide. The structure establishes the molecular mechanism underlying CRBN's enantioselective action. Further structure–function analysis reveals that these drugs have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins.

The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner 1 – 3 . LAT1, an antiporter of the amino acid–polyamine–organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as l -3,4-dihydroxyphenylalanine across membranes 2 – 6 . Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs 7 – 11 . LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)—a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane 8 , 9 . Despite extensive cell-based characterization of the LAT1–4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown 12 – 19 . Here we report the cryo-electron microscopy structures of human LAT1–4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1–4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease. The cryo-EM structure of the LAT1–4F2hc complex, a heteromeric amino acid transporter, is characterized, providing insights into its function and the mechanisms through which mutations of this complex could cause disease.

A protein complex composed of KPTN, ITFG2, C12orf66 and SZT2, named KICSTOR, is necessary for lysosomal localization of GATOR1, interaction of GATOR1 with the Rag GTPases and GATOR2, and nutrient-dependent mTORC1 modulation. KICSTOR is a negative regulator of mTORC1 signalling The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and organismal homeostasis and is deregulated in many human diseases, including epilepsy and cancer. In response to nutrients, mTORC1 is recruited to the lysosome by the Rag family of GTPases, whose activity is regulated by the GATOR complex. Here David Sabatini and colleagues identify a four-membered protein complex that they term KICSTOR. It localizes to lysosomes and interacts with GATOR to negatively regulate the pathway through which mTORC1 senses nutrients. In mice lacking one of the KICSTOR subunits, SZT2, mTORC1 signalling is hyperactivated in several tissues. A related paper in this week's issue of Nature from Ming Li and colleagues identifies the protein SZT2 as a negative regulator of mTORC1 signalling. Together, the two papers offer insight into mTORC1 regulation at the lysosome and could have implications for diseases associated with hyperactive mTORC1 signalling. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy 1 , 2 , 3 . Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation 4 . Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling 5 , 6 , 7 , 8 , 9 , 10 . Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease 11 , 12 .