Autosomal-dominant polycystic kidney disease (ADPKD) is primarily of adult-onset and caused by pathogenic variants in PKD1 or PKD2. Yet, disease expression is highly variable and includes very early-onset PKD presentations in utero or infancy. In animal models, the RNA-binding molecule Bicc1 has been shown to play a crucial role in the pathogenesis of PKD. To study the interaction between BICC1, PKD1, and PKD2, we combined biochemical approaches, knockout studies in mice and Xenopus, genetic engineered human kidney cells carrying BICC1 variants, as well as genetic studies in a large ADPKD cohort. We first demonstrated that BICC1 physically binds to the proteins Polycystin-1 and -2 encoded by PKD1 and PKD2 via distinct protein domains. Furthermore, PKD was aggravated in loss-of-function studies in Xenopus and mouse models, resulting in more severe disease when Bicc1 was depleted in conjunction with Pkd1 or Pkd2. Finally, in a large human patient cohort, we identified a sibling pair with a homozygous BICC1 variant and patients with very early onset PKD (VEO-PKD) that exhibited compound heterozygosity of BICC1 in conjunction with PKD1 and PKD2 variants. Genome editing demonstrated that these BICC1 variants were hypomorphic in nature and impacted disease-relevant signaling pathways. These findings support the hypothesis that BICC1 cooperates functionally with PKD1 and PKD2, and that BICC1 variants may aggravate PKD severity, highlighting RNA metabolism as an important new concept for disease modification in ADPKD.

Mosses from the genus Sphagnum have experienced 350 million years of evolution separate from all other mosses, resulting in distinctive features such as unlimited apical growth potential which is not seen in other mosses. Sphagnum mosses are ecosystem engineers and the main components of peatlands. Although peatlands cover only a small part of Earth’s landmass, they store more carbon than all living matter combined. Peat mining and agriculture result in degraded peatlands and thus have a dramatic negative impact on our climate. Sphagnum farming is a promising approach to combat climate change. Here, we review state of the art research with a focus on the establishment of a peat moss collection and their growth in vitro, especially in photobioreactors. Axenic, monoclonal Sphagnum strains have been established from spore capsules that have been collected from different peatlands across Europe. Analyses with flow cytometry have revealed haploid as well as diploid accessions. Optimization of the media composition for several species has resulted in an up to 50-fold biomass increase in a photobioreactor process. Future work should employ transcriptomics for further optimization of biomass gain. Moreover, the importance of the Sphagnum microbiome should be considered for transplantation of the axenic, clonal moss material to open fields.

Scalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals. Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen-producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions, including CO2 supplementation, pH, and light intensity, were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements.

The evolutionary conserved TOR kinase positively controls growth and development of the moss Physcomitrella, development and function of its chloroplasts, its protein synthesis, and cell-cycle progression. Target of Rapamycin (TOR) is a conserved protein kinase and an important signalling hub in eukaryotes. The moss Physcomitrella (Physcomitrium patens) is a model organism for plant physiology, development, and evolution. However, little is known about TOR signalling in non-vascular plants, including Physcomitrella. Here, we report the effects of inhibiting TOR signalling in Physcomitrella. We identified and characterised Physcomitrella 12-kDa FK506-binding protein (FKBP12), which binds TOR in the presence of rapamycin. Whereas the growth of wild-type protonema is unaffected by rapamycin, overexpressing endogenous FKBP12 rendered the plant susceptible to the inhibitor in a dose-dependent manner. Likewise, protonema growth was inhibited when the TOR-specific inhibitor AZD8055 was present in the culture. We show that rapamycin and AZD8055 have pleiotropic effects, as they delay cell-cycle progression and development, induce chlorosis, inhibit photosynthesis, and alter total protein content. Additionally, we identified and characterised PpTOR, PpLST8, and PpRAPTOR, key components of TOR complex 1 (TORC1), whereas RICTOR and mSIN1 of TORC2 are not encoded by the P. patens genome. We found that PpLST8 substitutes its homolog in yeast to allow cell growth. Physcomitrella mutants were generated with a conditional downregulation of PpTOR, PpLST8, and PpRAPTOR, respectively. They were impaired in growth. Finally, we show TOR-dependent phosphorylation of a well-known TOR phospho-target, the ribosomal protein RPS6, via LC–MS/MS. Collectively, our results show that Physcomitrella growth and development is positively controlled by a conserved TOR kinase. We suggest to further dissect TOR signalling in Physcomitrella in order to elucidate signalling integration via TORC1 in plants.

We report the successful moss-produced recombinant spider silk key protein component containing both the N- and the C-terminal domain. Spider dragline silk stands out as a remarkable biomaterial, representing one of nature's toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, the protein LhMaSp1, in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N- and C-terminal domains of LhMaSp1 in moss cells. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. We estimate that the yield of recombinant spider silk protein in Physcomitrella bioreactors is above 0.82 mg/g fresh weight.

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement, and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet α-granules and released from activated endothelial cells, interacts with factor H (FH) and vWF. However, to our knowledge, direct regulatory interaction with the complement cascade has not yet been described. Our study shows that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins and inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. We validated complement-regulatory function in blood samples from patients with primary complement defects. The physiological relevance of TSP-1 was demonstrated in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis after TSP-1 deficiency in an in vitro and in vivo model of AAV. The complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia and complement. In particular, the interplay between released TSP-1 and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

Bacterial cell division and plant chloroplast division require selfassembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO 2 supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. Scalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals.

Human Papilloma Virus (HPV) infections are the main causal agent of cervical and other anogenital cancers. Modern HPV vaccines are based on virus-like particles (VLPs), which are self-assembling nanoparticles composed of viral structural proteins. VLPs mimic native virions but lack viral DNA, rendering these pseudovirions non-infectious while retaining high immunogenicity. Despite available vaccines, the majority of cervical cancer deaths are due to HPV infections, particularly in low- and middle-income countries where inequalities in access to vaccination, screening, and treatment persist. While current HPV vaccines are produced in yeast or insect cells, alternative production platforms are explored, focusing on cost-effectiveness and scalability. Here, we used the moss plant Physcomitrella (Physcomitrium patens) to produce HPV-16 VLPs via expression of the major structural protein L1. We characterized Physcomitrella chloroplast transit peptides, which we used for targeting of L1 to chloroplasts, leading to higher recombinant protein accumulation compared to nuclear or cytoplasmic localization. We confirmed subcellular localization with confocal laser scanning microscopy and found L1 to accumulate within the chloroplast stroma. Production in 5 L-photobioreactors yielded more than 0.3 mg L1 per g fresh weight. We established a purification protocol for moss-produced L1 using a combination of ammonium sulphate precipitation and cation exchange chromatography. Purified samples were subjected to a controlled dis- and reassembly, ultimately yielding fully assembled HPV-16 L1 VLPs. This is the first report of production, purification and assembly of pseudovirions in a non-vascular plant. ### Competing Interest Statement The authors have declared no competing interest.

Target of rapamycin (TOR) is a conserved protein kinase and an important signalling hub in eukaryotes. The moss Physcomitrella ( Physcomitrium patens ) is a model organism for plant physiology, development, and evolution. However, little is known about TOR signalling in non-vascular plants, including Physcomitrella. Here, we report the effects of inhibiting TOR signalling in Physcomitrella. We identified and characterized Physcomitrella 12-kD FK506-binding protein (FKBP12), which binds TOR in the presence of rapamycin. Whereas the growth of wild-type protonema is unaffected by rapamycin, overexpressing endogenous FKBP12 rendered the plant susceptible to the inhibitor in a dose-dependent manner. Likewise, protonema growth was inhibited when the TOR-specific ATP-competitive inhibitor AZD8055 was present in the culture. We show that rapamycin and AZD8055 have pleiotropic effects, as they delay cell cycle progression and development, induce chlorosis, inhibit photosynthesis, and alter total protein content. Additionally, we identified and characterized PpTOR, PpLST8, and PpRAPTOR, key components of TOR complex 1 (TORC1), whereas RICTOR and mSIN1 of TORC2 are not encoded by the moss genome. We found that PpLst8 substitutes its homolog in yeast to allow cell growth. Physcomitrella mutants were generated with a conditional downregulation of PpTOR , PpLST8 , and PpRAPTOR , respectively. They were impaired in growth. Finally, we show TOR-dependent phosphorylation of a well-known TOR phospho-target, the ribosomal protein RPS6, via LC-MS/MS. Collectively, our results show that Physcomitrella growth and development is positively controlled by a conserved TOR kinase. We suggest to further dissect TOR signalling in Physcomitrella in order to elucidate signalling integration via TORC1 in plants. Key message The evolutionary conserved TOR kinase positively controls growth and development of the moss Physcomitrella, development and function of its chloroplasts, its protein synthesis and cell cycle progression. ### Competing Interest Statement The authors have declared no competing interest. European Union, 101065000 Deutsche Forschungsgemeinschaft, CIBSS - EXC-2189 - Project ID 390939984

First production of virus-like particles as a vaccine candidate in a non-vascular plant. Virus-like particles (VLPs) are self-assembling nanoparticles composed of viral structural proteins which mimic native virions but lack viral DNA and infectivity. VLPs are a resourceful class of biopharmaceuticals applied as subunit vaccines or as delivery vehicles for drugs and nucleic acids. Similar to viruses, VLPs are diverse in structure, composition, and assembly, requiring a tailored production platform aligned with the intended application. The moss plant Physcomitrella (Physcomitrium patens) is an emerging expression system offering humanized N-glycosylation, scalability, and adaptability to existing industry settings. Here, we used Physcomitrella to produce human papillomavirus (HPV) 16 VLPs. HPV VLPs are composed of the major structural protein L1 and are used as vaccines against HPV infections which are the main causal agent of cervical and other anogenital cancers. We characterized Physcomitrella chloroplast transit peptides, which we used for targeting of moss-produced L1 to chloroplasts, leading to higher recombinant protein yield compared to nuclear or cytoplasmic localization. We confirmed subcellular localization with confocal laser scanning microscopy and found L1 to accumulate within the chloroplast stroma. Production in 5-L photobioreactors yielded over 0.3 mg L1 per gram fresh weight. We established a purification protocol for moss-produced L1 using a combination of ammonium sulphate precipitation and cation exchange chromatography. Purified samples were subjected to a controlled dis- and reassembly, yielding fully assembled HPV-16 L1 VLPs. This is the first report of production, purification, and assembly of VLPs in a non-vascular plant.

Collagens are important structural proteins of the extracellular matrix that are essential for skin elasticity and integrity and are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via PEG-mediated protoplast transformation. Immunodetection identified collagen polypeptide-producing lines, and mass spectrometric analysis validated the product and confirmed its prolyl-hydroxylation. The presence of this important post-translational modification underscores the high biomimetic quality of the product derived from moss. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO2 supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. Key message Moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen, offering a sustainable and vegan alternative to conventional collagens derived from animals. ### Competing Interest Statement G.G. and B.H. are employees of Mibelle Biochemistry, which will bring moss-made human collagen to the market. All other authors declare no conflict of interest. Deutsche Forschungsgemeinschaft, CIBSS – EXC-2189 – Project ID 390939984

Spider dragline silk stands out as a remarkable biomaterial, representing one of nature's toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, (LhMaSp1) in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N- and C-terminal domains (NTD and CTD) of LhMaSp1 protein in moss cells. NTD and CTD are necessary domains for protein assembly in spider silk. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. ### Competing Interest Statement The authors have declared no competing interest.

Key message Characterization of Physcomitrella 3′UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. Abstract The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella ( Physcomitrium patens ) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3′UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3′UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3′UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3′UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

Cell division in bacteria and plastid division in plants both require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins as key components of their division machinery. FtsZ proteins are soluble GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo . We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli , which led to the formation of defined structures within the cells and to an influence on bacterial cell division. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 from E. coli and characterized their GTPase activity and polymerization in vitro . Both FtsZ isoforms showed GTPase activity, a prerequisite for polymerization. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2, but not of FtsZ2-1. In contrast, transmission electron microscopy demonstrated GTP-dependent filament formation of both isoforms. Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different and that both isoforms differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants. ### Competing Interest Statement The authors have declared no competing interest.

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement and platelets in these conditions are only partially understood.Thrombospondin-1 (TSP-1), found in platelet α-granules and released from activated endothelial cells, interacts with factor H (FH) and von Willebrand factor (vWF). However, direct regulatory interaction with the complement cascade has not yet been described. We could show that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins, inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. Complement-regulatory function is validated in blood samples from patients with primary complement defects. Physiological relevance of TSP-1 is demonstrated in ANCA-vasculitis patients by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis following TSP-1 deficiency in an ANCA-vasculitis model. The newly described complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia, complement and platelets. In particular, the interplay between released TSP-1, vWF and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases. ### Competing Interest Statement The authors have declared no competing interest.

Introduction:Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney disease in children. Although only 20% of cases can be genetically explained, the majority remain without an identified underlying etiology. The neurodevelopmental disorder Chung-Jansen syndrome (CHUJANS) is caused by haploinsufficiency of Pleckstrin homology domain-interacting protein (PHIP) and was previously associated with genital malformations. Anecdotal coincidence of CHUJANS and CAKUT prompted us to investigate whether urorenal malformations are part of the phenotypic spectrum of CHUJANS. Methods:Analysis of existing CHUJANS and CAKUT cohorts, consulting matchmaking platforms, and systematic literature review to look for additional patients with both CHUJANS and CAKUT. Prenatal expression studies in murine and human renal tissues to investigate the role for PHIP in kidney development. Results:We identified 4 novel and 8 published cases, indicating variable expressivity with a urorenogenital trait frequency of 5% to 35%. The prenatal expression studies supported a role for PHIP in normal kidney and urinary tract development. Conclusion:Pathogenic PHIP gene variants should be considered as causative in patients with syndromal CAKUT. Conversely, patients with CHUJANS should be clinically evaluated for urorenogenital manifestations. Because neurodevelopmental disorders are often associated with kidney phenotypes, an interdisciplinary re-evaluation offers promise in identifying incompletely penetrant kidney associations and uncovering novel molecular mechanisms of disturbed nephrogenesis.

1.AbstractBacterial cell division and plant chloroplast division require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoformsviaco-immunoprecipitation and mass spectrometry, and found protein-protein interactionin vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both inE. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed inE. coliand characterized their GTPase activity and polymerizationin vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interactin vivoandin vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

Hydroxylation of prolines to 4-trans-hydroxyproline (Hyp) is mediated by prolyl 4-hydroxylases (P4Hs). In plants, Hyps occur in Hydroxyproline-rich glycoproteins (HRGPs), and are frequently O-glycosylated. While both modifications are important, e.g. for cell wall stability, they are undesired in plant-made pharmaceuticals. Sequence motifs for prolyl-hydroxylation were proposed but did not include data from mosses, such as Physcomitrella. We identified six moss P4Hs by phylogenetic reconstruction. Our analysis of 73 Hyps in 24 secretory proteins from multiple mass spectrometry datasets revealed that prolines near other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95% of Hyps were predictable with combined established methods. In our data, AOV was the most frequent pattern. A combination of 443 AlphaFold models and MS data with 3000 prolines found Hyps mainly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a p4h1 knock-out (KO) Physcomitrella mutant. Quantitative proteomics with different p4h mutants revealed specific changes in protein amounts, and a modified prolyl-hydroxylation pattern, suggesting a differential function of the Physcomitrella P4Hs. Quantitative RT-PCR revealed a differential effect of single p4h KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 suggested significant amino acids in the active centre of the enzyme and revealed differences between P4H1 and the other Physcomitrella P4Hs.