Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various ...IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.
Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it ...can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1-10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.
Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase that promotes viral integration by tethering the preintegration complex to the chromatin. By virtue of its ...crucial role in the early steps of HIV replication, the interaction between LEDGF/p75 and integrase represents an attractive target for antiviral therapy. We have rationally designed a series of 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) that act as potent inhibitors of the LEDGF/p75-integrase interaction and HIV-1 replication at submicromolar concentration by blocking the integration step. A 1.84-A resolution crystal structure corroborates the binding of the inhibitor in the LEDGF/p75-binding pocket of integrase. Together with the lack of cross-resistance with two clinical integrase inhibitors, these findings define the 2-(quinolin-3-yl)acetic acid derivatives as the first genuine allosteric HIV-1 integrase inhibitors. Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor.
Molecular and protein biomarker profiling are key to oncology drug development. Antibody-drug conjugates (ADCs) directly deliver chemotherapeutic agents into tumor cells based on unique cancer cell ...biomarkers. A pan-cancer tissue microarray (TMA) data set and gene panel were validated and gene signature analyses were conducted on normal and cancer tissues to refine selection of ADC targets. Correlation of mRNA and protein levels, and human epidermal growth factor receptor (HER) expression patterns were assessed. An EdgeSeq biomarker panel (2862 genes) was used across 8531 samples (23 solid cancer types/subtypes; 16 normal tissues) with an established TMA data set, and immune cell and cell cycle gene signatures were analyzed. Discriminating gene expression signatures were defined based on pathological classification of cancer subtypes. Correlative analyses of HER2 and HER3 mRNA (EdgeSeq) and protein expression (immunohistochemistry IHC) were performed and compared with publicly available data (The Cancer Genome Atlas TCGA; Cancer Cell Line Encyclopedia CCLE). Gene expression patterns among cancer types in the TMA (EdgeSeq) and TCGA (RNA-seq) were similar. EdgeSeq gene signature analyses aligned with the majority of pathological cancer types/subtypes and identified cancer-specific gene expression patterns. TMA IHC H-scores for HER3 varied across cancer types/subtypes. In a few cancer types, HER3 mRNA and protein expression did not align, including lower liver hepatocellular carcinoma IHC H-score, compared with mRNA. Although all TNBC and ovarian cancer subtypes expressed mRNA, some had lower protein expression. This was seen in TMA and TCGA data sets, but not in CCLE. The EdgeSeq TMA data set can expand upon current biomarker data by including cancers not currently in TCGA. The primary analysis of EdgeSeq and IHC comparison suggested a unique protein-level regulation of HER3 in some tumor subtypes and highlights the importance of investigating protein levels of ADC targets in both tumor and normal tissues.
Because all regions of the plasmid share a strong identity with plasmids isolated from colistin-sensitive strains, it is unclear how novel resistance determinants are encoded.
Intermediate filaments (IFs) are essential cytoskeletal components in metazoan cells. They assemble from elementary dimers that are built around the central α-helical coiled-coil rod domain ...representing the IF ‘signature’. The rod consists of two similarly-sized parts, coil 1 and coil 2, connected by a non-α-helical linker L12. Coil 2 is absolutely conserved in length across all IF types and was initially predicted to consist of a short coiled-coil segment 2A based on a heptad pattern of hydrophobic residues, another linker L2 and a coiled-coil segment 2B. Here we present the crystal structure of human vimentin fragment including residues 261–335 i.e. approximately the first half of coil 2. The N-terminal part of this fragment reveals a parallel α-helical bundle characterized by 3.5 consecutive hendecad repeats. It is immediately followed by a regular left-handed coiled coil. The distinct non-helical linker L2 is therefore not observed. Together with the previously determined crystal structure of the major part of segment 2B (Strelkov et al., 2002), we can now build a complete atomic model of the 21nm long vimentin coil 2 dimer being a relatively rigid rod.
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. It has distinct molecular features and primarily affects the KIT and PDGFRA genes.
We ...wanted to assess the molecular profile of 68 GIST patients who were sequenced consecutively between 2014 and 2019 at our institute of pathology.
Our cohort comprised 60 primary and 8 metastatic GIST patients; 43 and 57% of the cases, respectively, were analyzed by Sanger sequencing or next-generation sequencing (NGS).
Of the 60 primary GIST patients, 47 (78%) showed a KIT mutation; 2 cases showed a double KIT mutation, and 1 of these was a therapy-naive GIST. Nine (15%) patients harbored a PDGFRA mutation, 2 (3%) had a BRAF mutation, 1 (2%) had a PIK3CA mutation, and 1 (2%) did not show any mutation. One BRAF and the PIK3CA mutation have not been described in GIST before. All metastatic GIST harbored exclusively KIT mutations.
A retrospective analysis of GIST sequenced at our institute revealed incidences of KIT and PDGFRA mutations comparable to those in other cohorts from Europe. Interestingly, we found 2 previously undescribed mutations in the BRAF and PIK3CA genes as well as 1 treatment-naive case with a double KIT mutation in exon 11.
The development of resistance to virtually all current antibiotics makes the discovery of new antimicrobial compounds with novel protein targets an urgent challenge. The dapE-encoded ...N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is an essential metallo-enzyme for growth and proliferation in many bacteria, acting in the desuccinylation of N-succinyl-L,L-diaminopimelic acid (SDAP) in a late stage of the anabolic pathway towards both lysine and a crucial building block of the peptidoglycan cell wall. L-Captopril, which has been shown to exhibit very promising inhibitory activity in vitro against DapE and has attractive drug-like properties, nevertheless does not target DapE in bacteria effectively. Here we show that L-captopril targets only the Zn(2+)-metallo-isoform of the enzyme, whereas the Mn(2+)-enzyme, which is also a physiologically relevant isoform in bacteria, is not inhibited. Our finding provides a rationale for the failure of this promising lead-compound to exhibit any significant antibiotic activity in bacteria and underlines the importance of addressing metallo-isoform heterogeneity in future drug design. Moreover, to our knowledge, this is the first example of metallo-isoform heterogeneity in vivo that provides an evolutionary advantage to bacteria upon drug-challenge.
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