Max Planck Institute of Biochemistry

Kardell O., Gronauer T., von Toerne C., Merl-Pham J., König A., Barth T.K., Mergner J., Ludwig C., Tüshaus J., Giesbertz P., Breimann S., Schweizer L., Müller T., Kliewer G., Distler U., et. al.

Kabatnik S., Zheng X., Pappas G., Steigerwald S., Padula M.P., Mann M.

Abstract Signet Ring Cell Carcinoma (SRCC) is a rare and highly malignant form of adenocarcinoma with increasing incidence and poor prognosis due to late diagnosis and limited treatment options. We employed Deep Visual Proteomics (DVP), which combines AI-directed cell segmentation and classification with laser microdissection and ultra-high sensitivity mass spectrometry, for cell-type-specific proteomic analysis of SRCC across the bladder, prostate, seminal vesicle, and a lymph node of a single patient. DVP identified significant alterations in DNA damage response (DDR) proteins, particularly within the ATR and mismatch repair (MMR) pathways, indicating replication stress as a crucial factor in SRCC mutagenicity. Additionally, we observed substantial enrichment of immune-related proteins, reflecting high levels of cytotoxic T lymphocyte infiltration and elevated PD-1 expression. These findings suggest that pembrolizumab immunotherapy may be more effective than conventional chemotherapy for this patient. Our results provide novel insights into the proteomic landscape of SRCC, identify potential targets, and open up for personalized therapeutic strategies in managing SRCC.

Garriga F., Martínez-Hernández J., Parra-Balaguer A., Llavanera M., Yeste M.

Liquid storage is the primary preservation method in the swine breeding industry because of its advantages over cryopreservation. Calcium (Ca2+), a key regulator of cell physiology, plays a crucial role during liquid preservation. Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPases (SERCA) belong to a family of P-type ATPases that regulate Ca2+ homeostasis within cells and have been previously described to play a function in the sperm of various mammalian species. Herein, we hypothesized that SERCA2 is present in pig sperm and is involved in the resilience of this cell to liquid preservation at 17 °C. For this purpose, sperm were incubated with different concentrations of thapsigargin (Thg; 0, 5, 25, and 50 µM) and stored at 17 °C for ten days. The presence and localization of SERCA2 were evaluated using immunoblotting and immunofluorescence, respectively. On days 0, 4, and 10, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system, and sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), and intracellular levels of Ca2+, superoxides and total reactive oxygen species (ROS) were evaluated by flow cytometry. We localized SERCA2 in the acrosome and midpiece of pig sperm. Furthermore, inhibition of SERCA with Thg resulted in reduced sperm viability and membrane stability, and increased MMP, and Ca2+ and ROS levels. In conclusion, the activity of SERCA prevents the accumulation of intracellular Ca2+ in sperm, which is detrimental to sperm quality and function during liquid storage at 17 °C. We thus suggest that the function of SERCA is crucial for the preservation of pig semen.

Guinart A., Doellerer D., Qutbuddin Y., Zivkovic H., Branca C., Hrebik D., Schwille P., Feringa B.L.

Li Y., Chen D., Tang T., Shen X.

We explore the application of Vision Transformer (ViT) for handwritten text recognition. The limited availability of labeled data in this domain poses challenges for achieving high performance solely relying on ViT. Previous transformer-based models required external data or extensive pre-training on large datasets to excel. To address this limitation, we introduce a data-efficient ViT method that uses only the encoder of the standard transformer. We find that incorporate a Convolutional Neural Network (CNN) for feature extraction instead of the original patch embedding and employ Sharpness-Aware Minimization (SAM) optimizer to ensure that the model can converge towards flatter minima yield notable enhancements. Furthermore, our introduction of the span mask technique, which masks interconnected features in the feature map, acts as an effective regularizer. Empirically, our approach competes favarably with traditional CNN-based models on small datasets like IAM and READ2016. Additionally, it establishes a new benchmark on the LAM dataset, currently the largest dataset with 19,830 training text lines. The code will be publicly available at: https://github.com/YutingLi0606/HTR-VT.

Chakraborty S., Martinez-Sanchez A., Beck F., Toro-Nahuelpan M., Hwang I., Noh K., Baumeister W., Mahamid J.

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) organization and structures of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell–derived neurons, and pluripotent and differentiated P19 cells. We obtain in situ density maps of several lumenal particles from the respective cells and detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggest that a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggests a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.

Barrera-Paez J.D., Bacman S.R., Balla T., Van Booven D., Gannamedi D.P., Stewart J.B., Mok B., Liu D.R., Lombard D.B., Griswold A.J., Nedialkova D.D., Moraes C.T.

Primary mitochondrial disorders are most often caused by deleterious mutations in the mitochondrial DNA (mtDNA). Here, we used a mitochondrial DddA-derived cytosine base editor (DdCBE) to introduce a compensatory edit in a mouse model that carries the pathological mutation in the mitochondrial transfer RNA (tRNA) alanine (mt-tRNA Ala ) gene. Because the original m.5024C→T mutation (G→A in the mt-tRNA Ala ) destabilizes the mt-tRNA Ala aminoacyl stem, we designed a compensatory m.5081G→A edit (C→T in the mt-tRNA Ala ) that could restore the secondary structure of the tRNA Ala aminoacyl stem. For this, the DdCBE gene construct was initially tested in an m.5024C→T mutant cell line. The reduced mt-tRNA Ala amounts in these cells were increased after editing up to 78% of the mtDNA. Then, DdCBE was packaged in recombinant adeno-associated virus 9 (AAV9) and intravenously administered by retro-orbital injections into mice. Expression of the transduced DdCBE was observed in the heart and skeletal muscle. Total mt-tRNA Ala amounts were restored in heart and muscle by the m.5081G→A edit in a dose-dependent manner. Lactate amounts, which were increased in the heart, were also decreased in treated mice. However, the highest dose tested of AAV9-DdCBE also induced severe adverse effects in vivo because of the extensive mtDNA off-target editing that it generated. These results show that although DdCBE is a promising gene therapy tool for mitochondrial disorders, the doses of the therapeutic constructs must be carefully monitored to avoid deleterious off-target editing.

Muhar M.F., Farnung J., Cernakova M., Hofmann R., Henneberg L.T., Pfleiderer M.M., Denoth-Lippuner A., Kalčic F., Nievergelt A.S., Peters Al-Bayati M., Sidiropoulos N.D., Beier V., Mann M., Jessberger S., Jinek M., et. al.

Abstract During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts1. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites2,3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins3–6; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1–CUL1–F-box protein (SCF) ubiquitin ligase SCF–FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

Kraus F., He Y., Swarup S., Overmyer K.A., Jiang Y., Brenner J., Capitanio C., Bieber A., Jen A., Nightingale N.M., Anderson B.J., Lee C., Paulo J.A., Smith I.R., Plitzko J.M., et. al.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1 −/− and NPC2 −/− mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo–electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2 −/− cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

Ogger P.P.

Skowronek P., Wallmann G., Wahle M., Willems S., Mann M.

Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms; however, it is challenging to implement in routine settings. In this protocol, we combine a robust chromatographic platform with a high-performance mass spectrometric setup to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients (Evosep One) combined with a trapped ion mobility time-of-flight mass spectrometer (timsTOF, Bruker). The timsTOF enables parallel accumulation–serial fragmentation (PASEF), in which ions are accumulated and separated by their ion mobility, maximizing ion usage and simplifying spectra. Combined with data-independent acquisition (DIA), it offers high peak sampling rates and near-complete ion coverage. Here, we explain how to balance quantitative accuracy, specificity, proteome coverage and sensitivity by choosing the best PASEF and DIA method parameters. The protocol describes how to set up the liquid chromatography–mass spectrometry system and enables PASEF method generation and evaluation for varied samples by using the py_diAID tool to optimally position isolation windows in the mass-to-charge and ion mobility space. Biological projects (e.g., triplicate proteome analysis in two conditions) can be performed in 3 d with ~3 h of hands-on time and minimal marginal cost. This results in reproducible quantification of 7,000 proteins in a human cancer cell line in quadruplicate 21-min injections and 29,000 phosphosites for phospho-enriched quadruplicates. Synchro-PASEF, a highly efficient, specific and novel scan mode, can be analyzed by Spectronaut or AlphaDIA, resulting in superior quantitative reproducibility because of its high sampling efficiency. Aligning trapped ion mobility with a mass-selective quadrupole and time-of-flight mass spectrometry enables parallel accumulation–serial fragmentation, which improves proteomic analysis. This protocol comprehensively describes PASEF workflows.

Pousa S., Ramos-Bermúdez P.E., Besada V., Cabrales-Rico A., Guirola Cruz O., Garay H.E., Rodríguez-Mallón A., Zettl K., Wiśniewski J.R., González L.J.

Keyhole limpet haemocyanins (KLH1 and KLH2) from Megathura crenulata, are multi-subunit oxygen-carrying metalloproteins of approximately 3900 amino acids, that are widely used as carrier proteins in conjugate vaccines and in immunotherapy.

Egea‐Rodriguez S., Váraljai R., Nordmann T.M., Lubis R., Philip M., Rambow F., Roesch A., Flaig M., Horn S., Stoll R., Zhao F., Paschen A., Klebl B., Hickson I.D., Schadendorf D., et. al.

AbstractBackgroundCancer immunotherapy has transformed metastatic cancer treatment, yet challenges persist regarding therapeutic efficacy. RECQL4, a RecQ‐like helicase, plays a central role in DNA replication and repair as part of the DNA damage response, a pathway implicated in enhancing efficacy of immune checkpoint inhibitor (ICI) therapies. However, its role in patient response to ICI remains unclear.MethodsWe analysed whole exome and bulk RNA sequencing data from a pan‐cancer cohort of 25 775 patients and cutaneous melanoma cohorts (untreated: n = 471, anti‐progressive disease [PD]‐1 treated: n = 212). RECQL4 copy number variations and expression levels were assessed for patient outcomes. We performed gene set enrichment analysis to identify RECQL4‐dependent signalling pathways and explored the association between RECQL4 levels and immunoscores. We evaluated the interplay of ICI response and RECQL4 expression in melanoma cohorts of 95 responders and 85 non‐responders prior to and after ICI‐targeted therapy and tested the prognostic power of RECQL4. Finally, we generated genetically engineered RECQL4 variants and conducted comprehensive multi‐omic profiling, employing techniques such as liquid chromatography with tandem mass spectrometry, to elucidate mechanistic insights.ResultsWe identified RECQL4 as a critical negative regulator of poor prognosis and response to ICI therapy, but also demonstrated its suitability as an independent biomarker in melanoma. High tumour purity and limited signatures of tumour immunogenicity associated with response to anti‐PD‐1 correlated with high RECQL4 activity. We found alterations in the secretion profile of immune regulatory factors and immune‐related pathways robustly suppressed in tumours with high RECQL4 levels, underscoring its crucial role in fostering immune evasion. Mechanistically, we identified RECQL4‐mediated regulation of major histocompatibility complex class II molecule expression and uncovered class II major histocompatibility complex transactivator as a mediator bridging this regulation.ConclusionsOur findings unraveled the pivotal role of RECQL4 in immune modulation and its potential as both a predictive biomarker and therapeutic target for optimising immunotherapeutic strategies across various cancer types.Highlights High RECQL4 expression limits survival and can act as an independent prognostic factor in melanoma patients. RECQL4 has the potential to act as a negative feedback mediator of immune checkpoint‐targeted therapy by limiting signatures associated with therapeutic efficacy. RECQL4 favours an immune‐evasive phenotype by downregulating major histocompatibility complex class II molecules.

Xu R., Li K., Chen A., Chen Y., Sheng R., Chen C., Qin H., Zhang W., Liu Y., Rodríguez H.V., Kanaparti D., Zhu B.

ABSTRACTThe abandonment of cultivated fields notably affects soil characteristics. However, its impact on soil microbial community composition, particularly under different fertilization regimes, remains poorly understood. Therefore, we evaluated the effects of abandoned paddy fields with different fertilization histories (no fertilization, NK, NP, and NPK) on the abundance, composition, and their predicted function of soil bacteria and fungi. Our findings revealed that the abandonment led to a considerable decrease in soil bacterial and fungal biomass. Fertilization history and abandonment each significantly affected on the bacterial community, resulting in increased relative proportions of Acidobacteria and decreased relative proportions of Proteobacteria in the NP and NPK treatments compared to those in the CK and NK treatments. The fungal community only exhibited a notable response to abandonment, with variations in the relative abundance of Ascomycota, Mucoromycota, and Basidiomycota. Canonical correspondence analysis revealed the significant association between soil bacterial community and pH, total phosphorus, and available phosphorus, whereas the pH and NO3−–N were key drivers of fungal community variations in abandoned soils. Network analysis indicated that abandonment enhanced the complexity but decreased the stability of the bacterial community, while fungi exhibited the opposite trend. Our findings highlight that changes in bacterial functions caused by residual nutrients, particularly high residual phosphorus, may accelerate soil organic C decomposition. Overall, this study provides a novel insight into the shifts in soil bacterial and fungal communities and their ecological functions after the abandonment of cultivated fields.

Pegoraro C., Karpova E., Qutbuddin Y., Sanchis E.M., Dimitrijevs P., Huck‐Iriart C., Gavrilović S., Arsenyan P., Schwille P., Felip‐León C., Duro‐Castano A., Conejos‐Sanchez I., Vicent M.J.

AbstractMitochondria play critical roles in regulating cell fate, with dysfunction correlating with the development of multiple diseases, emphasizing the need for engineered nanomedicines that cross biological barriers. Said nanomedicines often target fluctuating mitochondrial properties and/or present inefficient/insufficient cytosolic delivery (resulting in poor overall activity), while many require complex synthetic procedures involving targeting residues (hindering clinical translation). The synthesis/characterization of polypeptide‐based cell penetrating diblock copolymers of poly‐L‐ornithine (PLO) and polyproline (PLP) (PLOn‐PLPm, n:m ratio 1:3) are described as mitochondria‐targeting nanocarriers. Synthesis involves a simple two‐step methodology based on N‐carboxyanhydride ring‐opening polymerization, with the scale‐up optimization using a “design of experiments” approach. The molecular mechanisms behind targetability and therapeutic activity are investigated through physical/biological processes for diblock copolymers themselves or as targeting moieties in a poly‐L‐glutamic (PGA)‐based conjugate. Diblock copolymers prompt rapid cell entry via energy‐independent mechanisms and recognize mitochondria through the mitochondria‐specific phospholipid cardiolipin (CL). Stimuli‐driven conditions and mitochondria polarization dynamics, which decrease efficacy depending on disease type/stage, do not compromise diblock copolymer uptake/targetability. Diblock copolymers exhibit inherent concentration‐dependent anti‐tumorigenic activity at the mitochondrial level. The diblock copolymer conjugate possesses improved safety, significant cell penetration, and mitochondrial accumulation via cardiolipin recognition. These findings may support the development of efficient and safe mitochondrial‐targeting nanomedicines.