Roman Sakson

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

The collapse of protein homeostasis manifests itself in a toxic protein aggregation cascade, which is associated with degenerative diseases and aging. To solubilize aggregates, dedicated protein disaggregases exist in unicellular organisms, but these have no nuclear/cytosolic orthologs in metazoa. Alternative metazoan disaggregation machines have been described, but how these are operated and regulated in vivo remained unknown. We show that protein disaggregases are functionally diversified in human cells to efficiently target different types of stress-induced aggregates in sequential and temporally distinct phases. In particular, we show the selective assembly of an Hsp70-DNAJA1-DNAJB1 trimeric disaggregase that forms during late phase of stress recovery., i.e. , after VCP-dependent solubilization of non-native proteins that accumulate in cellular condensates such as nucleoli or stress granules. When activated, the trimeric disaggregase provides resistance to stress toxicity and contributes to amyloid disposal. Strikingly, this disaggregase collapses early in cells undergoing replicative aging with important underlining pathophysiological consequences.

As novel liquid chromatography–mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36–60 μg result in optimal peptide recovery, but lower amounts ≥3 μg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

The current SARS-CoV-2/COVID-19 pandemic wreaks medical and socioeconomic havoc. Despite the availability of vaccines, cost-effective acute treatment options preventing morbidity and mortality are urgently needed. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 in therapeutic as well as prophylactic regimens. The herbal infusions exerted antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron.

Protein glycosylation is essential in all domains of life and its mutational impairment in humans can result in severe diseases named Congenital Disorders of Glycosylation (CDGs). Studies on molecular level are however challenging, because many glycosyltransferases in the endoplasmic reticulum (ER) are low abundance membrane proteins. We established a comprehensive multiple reaction monitoring (MRM) assay to quantify most human glycosyltransferases involved in the processes of N -glycosylation, O - and C -mannosylation in the ER. To increase reproducibility, a membrane protein fraction of isotopically labeled HEK 293T cells was used as an internal standard. With this internal standard the MRM assay is easily transferable between laboratories. 22 glycosyltransferases could be reliably quantified from whole cell lysates of HEK 293T cells, HeLa cells and skin fibroblast cell lines. We then analyzed fibroblasts derived from CDG type I patients with mutations in the ALG1 , ALG2 or ALG11 gene. Mutations in ALG1 or ALG2 gene strongly reduced the levels of the ALG1 and ALG2 protein, respectively. In contrast, the levels of all other glycosyltransferases remained unchanged, which was unexpected given evidence that the ALG1, ALG2 and ALG11 proteins form a stable complex. This study describes an efficient workflow for the development of MRM assays for low abundance proteins, establishes a ready-to-use tool for the comprehensive quantification of ER-localized glycosyltransferases and provides new insight into the organization of disease-relevant glycosylation processes.

Bacterial toxin–antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin–antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.