Multivalency of nucleosome recognition by LEDGF.

Eukaryotic transcription is dependent on specific histone modifications. Their recognition by chromatin readers triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. Here, we report the structures of a PWWP domain from transcriptional coactivator LEDGF in complex with the H3K36 di- and trimethylated nucleosome, indicating that both methylation marks are recognized by PWWP in a highly conserved manner. We identify a unique secondary interaction site for the PWWP domain at the interface between the acidic patch and nucleosomal DNA that might contribute to an H3K36-methylation independent role of LEDGF. We reveal DNA interacting motifs in the intrinsically disordered region of LEDGF that discriminate between the intra- or extranucleosomal DNA but remain dynamic in the context of dinucleosomes. The interplay between the LEDGF H3K36-methylation reader and protein binding module mediated by multivalent interactions of the intrinsically disordered linker with chromatin might help direct the elongation machinery to the vicinity of RNA polymerase II, thereby facilitating productive elongation.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy.

Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.

Cerebral small vessel disease-related protease HtrA1 processes latent TGF-ß binding protein 1 and facilitates TGF-ß signaling.

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor ß (TGF-ß) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-ß signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-ß pathway activation. We identify latent TGF-ß binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-ß bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-ß signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly.

The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason-Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.

Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.

The Impact of Lens Epithelium-Derived Growth Factor p75 Dimerization on Its Tethering Function.

The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration.

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1ß signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.

Conserved and variable features of Gag structure and arrangement in immature retrovirus particles.

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

A ubiquitous disordered protein interaction module orchestrates transcription elongation.

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

Molecular Mechanism of LEDGF/p75 Dimerization.

Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.

Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds.

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations.

Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.