Biology subjects

Rappsilber, J.

Publications and source records attributed to Rappsilber, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of Syn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-{beta} amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict Syn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

biophysics↗

bioRxiv2020

Cryo-EM reveals the complex architecture of dynactin's shoulder and pointed end

Dynactin is a 1.1 MDa complex that activates the molecular motor, dynein, for ultra-processive transport along microtubules. In order to do this it forms a tripartite complex with dynein and a coiled-coil adaptor. Dynactin consists of an actin-related filament whose length is defined by its flexible shoulder domain. Despite previous cryo-EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high-resolution information in these regions. Here we combine multiple cryo-EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder positions the four identical p50 subunits in different conformations to bind dynactins filament and securely houses the p150 subunit. The pointed end map allows us to build the first structure of p62, and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.

molecular biology↗

bioRxiv2020

The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19CCAN complex in meiotic kinetochore assembly

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganisation of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that replace the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that this kinetochore remodelling in meiosis requires the Ctf19cCCAN inner kinetochore complex. Through functional analyses, we identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth, but becomes critical upon meiotic entry. Therefore, extensive kinetochore remodelling and a distinct assembly pathway direct the specialization of meiotic kinetochores for successful gametogenesis.

cell biology↗

bioRxiv2020

Isolation of protein N-terminal peptides by charge-mediated position- elective enrichment using strong cation exchange chromatography

We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N-termini, and then the N-terminal peptides are separated from the internal peptides by means of CHArge-Mediated Position-selective (CHAMP) enrichment using strong cation exchange (SCX) chromatography. This CHAMP-SCX approach was applied to 20 g of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1,550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N-termini registered in the Swiss-Prot database. Since this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N-termini, including proteoforms with neo-N-termini.

cell biology↗

bioRxiv2020

Reliable identification of protein-protein interactions by crosslinking mass spectrometry

Crosslinking mass spectrometry is widening its scope from structural analyzes of purified multi-protein complexes towards systems-wide analyzes of protein-protein interactions. Assessing the error in these large datasets is currently a challenge. Using a controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate a reliable false-discovery rate estimation procedure for protein-protein interactions identified by crosslinking mass spectrometry.

systems biology↗

bioRxiv2020

Stress-induced translation inhibition through rapid displacement of scanning initiation factors

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA-association. Among translation components, RNA-association was most reduced for initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5'-ends of mRNAs. Following glucose withdrawal, 5-binding was abolished within 30sec, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5 RNA-binding by initiation factors over [~]16min. Translation shutoff provoked selective 5'-degradation of mRNAs encoding translation-related factors, mediated by Xrn1. These results reveal mechanisms underlying translational control of gene expression during stress. HighlightsA quantitative proteomic approach reveals rapid stress-induced changes in RNA-binding Translation shutdown is driven by loss of mRNA binding by scanning initiation factors eIF4B and Ded1 have key but separate roles in driving the stress response Heat shock invokes rapid RNA degradation by Xrn1, selective for translation machinery

cell biology↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗

bioRxiv2020

Ultraviolet Photodissociation of tryptic peptide backbones at 213 nm

We analyzed the backbone fragmentation behavior of tryptic peptides of a four protein mixture and of E. coli lysate subjected to Ultraviolet Photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15,178 high-confidence peptide-spectrum matches by additionally recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor. Type a, b and y ions were most prominent in UVPD spectra and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity) and remaining precursor intensity high. Sequence coverage and sequence fragment intensity ratio correlated with precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to non-covalent interactions. UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points at aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange with identifier PXD018176 and on spectrumviewer.org/db/UVPD_213nm_trypPep.

systems biology↗

bioRxiv2020

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

molecular biology↗

bioRxiv2020

In-cell architecture of an actively transcribing-translating expressome

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

molecular biology↗

bioRxiv2020

The C-terminal helix of BubR1 is essential for CENP-E-dependent chromosome alignment

During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores, to facilitate chromosome alignment. The spindle checkpoint protein BubR1 has been reported as a CENP-E interacting partner, but to what extent, if at all, BubR1 contributes to CENP-E localization at kinetochores, has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, while a minimal key acidic patch on the kinetochore-targeting domain of CENP-E, is also essential. We then demonstrate that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. This BubR1-CENP-E axis is critical to align chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis.

cell biology↗

bioRxiv2020

Proteomics Using Protease Alternatives to Trypsin Benefits from Sequential Digestion with Trypsin

Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, pre-digesting with trypsin improves the number of identified proteins for proteinase K by 731 %. Trypsin pre-digestion also improved the protein identifications of other proteases, AspN (+62 %), GluC (+80 %) and chymotrypsin (+21 %). Interestingly, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.

biochemistry↗

bioRxiv2019

Hap2-Ino80 facilitated transcription promotes de novo establishment of CENP-A chromatin

Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily-conserved histone H3 variant, which directs kinetochore assembly and hence, centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity selected solubilised fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naive centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilises H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

molecular biology↗

bioRxiv2019

Architecture of the Mto1/2 microtubule nucleation complex

Proteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators of{gamma} -tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of the Schizosaccharomyces pombe CM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complex in vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimers in vitro and that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutants in vivo.

cell biology↗

bioRxiv2019

Structural Basis for CAL1-Mediated Centromere Maintenance

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting is achieved by HJURP and the Mis18 complex, respectively. Here, using crystal structures, we show how Drosophila CAL1, an evolutionarily distinct CENP-A chaperone, targets CENP-A to the centromere receptor CENP-C without the requirement of the Mis18 complex: while the N-terminal CAL1 fragment (CAL11-160) wraps around CENP-A/H4 through multiple physical contacts, the C-terminal CAL1 fragment (CAL1893-914) directly binds CENP-C cupin dimer. Our work shows CAL1, though divergent at the primary structure, employs evolutionarily conserved and adaptive structural principles to recognise CENP-A/H4 and CENP-C providing insights into the minimalistic principles underlying centromere maintenance.

biochemistry↗

bioRxiv2019

Laboratory evolution of Escherichia coli enables life based on fluorinated amino acids

Organofluorine compounds are toxic to various living beings in different habitats. On the other hand, fluorine incorporation into single proteins via related amino acid analogues has become common practice in protein engineering. Thus, an essential question remains: can fluorinated amino acids generally be used as xeno-nutrients to build up biomass, or do large amounts of fluorine in the cells render them nonviable? To gain information about the effect of long-term exposure of a cellular proteome to fluorinated organic compounds, we constructed an experiment based on bacterial adaptation in artificial fluorinated habitats. We propagated Escherichia coli (E. coli) in the presence of either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of tryptophan (Trp) analogues. We found that full adaptation requires astonishingly few genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity and quality control of protein folding. These findings highlight the cellular mechanisms of the evolutionary adaption process to unnatural amino acids and provide the molecular foundation for novel and innovative bioengineering of microbial strains with potential for biotechnological applications.\n\nOne Sentence SummaryLaboratory evolution enabled for the first time Escherichia coli to use fluorinated indoles as essential precursors for protein synthesis by introducing few genetic mutations but large rearrangements in regulatory networks, membrane integrity and quality control of protein folding.

synthetic biology↗

bioRxiv2019

Direct Nucleosome Binding of Borealin Secures Chromosome Association and Function of the Chromosomal Passenger complex

Chromosome association of the Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres [1-3] failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

cell biology↗

bioRxiv2019

The human proteome co-regulation map reveals functional relationships between proteins

The annotation of protein function is a longstanding challenge of cell biology that suffers from the sheer magnitude of the task. Here we present ProteomeHD, which documents the response of 10,323 human proteins to 294 biological perturbations, measured by isotope-labelling mass spectrometry. Using this data matrix and robust machine learning we create a co-regulation map of the cell that reflects functional associations between human proteins. The map identifies a functional context for many uncharacterized proteins, including microproteins that are difficult to study with traditional methods. Co-regulation also captures relationships between proteins which do not physically interact or co-localize. For example, co-regulation of the peroxisomal membrane protein PEX11{beta} with mitochondrial respiration factors led us to discover a novel organelle interface between peroxisomes and mitochondria in mammalian cells. The co-regulation map can be explored at www.proteomeHD.net.

systems biology↗