Abstract The stabilization of rhizobia in specialized organelles, called symbiosomes, is an evolutionary hallmark for maintaining high rates of nitrogen fixation in legumes. This is achieved by releasing thousands of bacteria from nodular infection threads within in a single plant cell, which poses a great challenge for the host to keep control over these differentiated bacteria. Considering the importance of proteolytic degradation of antimicrobial proteins or generation of symbiosis-promoting peptides, proteolytic activity may represent a key regulatory system. Indeed, we identified the Medicago truncatula subtilisin-like protease (subtilase, SBT) 12a acting as a novel regulator of symbiosome functionality and maintenance. Loss-of-function mutations in SBT12a led to severe symbiotic defects with nodules of sbt12a being characterized by high level induction of defense/senescence-related genes. Using untargeted proteomic High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) we identified and individually confirmed specific SBT12a target proteins that are involved in plant defense responses and symbiosome maintenance. This positions SBT12a as a central host factor controlling symbiosome performance.

Phytocytokines are endogenous peptides that modulate plant immunity outcomes, yet how their maturation and spatial deployment are controlled remains unclear. Here we show that the maize phytocytokine precursor PROZIP1 is controlled by a spatially separated, 2-stage proteolytic pathway that mechanistically uncouples signal activation from extracellular attenuation. PROZIP1 associates with the endoplasmic reticulum (ER) and undergoes intracellular, arginine-dependent processing by Type II metacaspases (MCAs), generating a C-terminal PROZIP1 fragment (Ct-PROZIP1). This processing licenses PROZIP1 for export to the apoplast via an ER-Golgi-independent route. Proteomic mapping and mutational analyses identify arginine residues flanking the Zip1 peptide as critical for efficient processing and secretion. The calcium-dependent MCA ZmMC9 specifically processes PROZIP1, thereby efficiently generating the bioactive Ct-PROZIP1 fragment. In the apoplast, Ct-PROZIP1 is further processed by papain-like cysteine proteases and additional extracellular proteases, contributing to Zip1 turnover and signal clearance. While the free Zip1 peptide is detected at later stages, Ct-PROZIP appears to be the primary signaling entity in modulating pathogen-induced immune responses. Together, these findings demonstrate a previously unknown complexity in peptide signaling, suggesting a multilayered control of phytocytokine activity that provides spatial and temporal precision to disease modulation in maize.

Plants perceive neighboring vegetation through an enrichment of far-red light (shade) in the environment. These changes in light quality trigger molecular and physiological responses aimed at outgrowing competitors, collectively known as the shade avoidance syndrome. In this study, we identify the TARGET OF RAPAMYCIN (TOR) complex 1 (TORC1), a major growth-regulating hub in eukaryotes, as a driver of shade-mediated growth responses in plants. Combining physiology, genetics, biochemistry, and proteomics, we show that TOR activity is rapidly enhanced upon shade perception and is required for proper shade responses, as TOR inhibition severely impairs shade-mediated elongation. Furthermore, we found that the control of shade-mediated elongation by TOR involves auxin-dependent mechanisms, requires efficient translation activity, and is closely linked to epidermal cell elongation capacity. Altogether, our work identifies TOR as a key integrator of light quality signals to control adaptive growth responses. Finally, we further highlight the conservation of shade-mediated TOR activation in tomato, with potential implications for engineering crop cultivars better suited to high-density planting.

ABSTRACT In contrast to their important structural and regulatory functions, such as in the metabolism of cyanobacteria, genes encoding small proteins are often not well characterized. Cyanobacteria use redox equivalents and energy from oxygenic photosynthesis to produce organic carbon compounds from inorganic carbon (C i ) and organic nitrogen compounds from inorganic nitrogen sources. Therefore, the assimilation and metabolism of carbon and nitrogen are coordinated at multiple levels in cyanobacteria. Here, we analyzed the Synechocystis sp. PCC 6803 gene ssr3189 encoding a 55 amino acids protein. Orthologs were detected in 665 cyanobacterial genomes defining COG5794 in the Database of Clusters of Orthologous Genes. Homologs in several eukaryotic algae suggest that Ssr3189 is an important protein that originated in cyanobacteria, was retained in algae after endosymbiosis, but was lost in plants. Polynucleotide kinase assays validated Ssr3189 as an RNA-binding protein. Deletion of ssr3189 resulted in lower pigmentation, delayed growth, and alterations in the expression of genes encoding transporters for nitrogen and C i , and metabolic enzymes. Metabolomic analysis revealed a substantial overaccumulation of glutamine and tricarboxylic acid cycle intermediates in the deletion mutant, and further differences in the amino acid and organic acid pools compared to the wild type. Co-immunoprecipitation analysis yielded ribosomal protein S21, enolase and the Cas6-1 endoribonuclease as the most strongly co-enriched proteins, together with all other ribosomal proteins and a small set of metabolic enzymes. These findings are consistent with observations that ssr3189 encodes the ribosome-associated protein cS24 and suggest that it connects translation with metabolic control, and, potentially, RNA decay. IMPACT STATEMENT Despite considerable progress in analyzing microbial genomes, there are still substantial numbers of uncharacterized gene functions. Here, we analyzed a mutant lacking gene ssr3189 that is widely conserved, but phenotypically uncharacterized in cyanobacteria. This gene is important for growth at the optimum temperature and essential at lower temperatures. In its absence, important metabolites were overaccumulated, while genes involved in nitrogen and C i uptake were dysregulated. The encoded protein binds RNA and interacts with proteins involved in translation and metabolism. The findings are consistent with a function as a ribosomal protein bridging protein synthesis and the regulation of metabolism.

In a previous study, we discovered that Litorilinea aerophila, a member of the bacterial phylum Chloroflexota, had acquired a bona fide archaellum gene cluster through horizontal gene transfer from Archaea, a surprising finding given that the archaellum had long been considered an archaeal-specific motility machinery. Here, we hypothesize that the distinctive multilayered cell envelope of L. aerophila provides the structural context that enables the integration and function of the archaellum motility machinery. Using fluorescence microscopy, thin-section electron microscopy, and cryo-electron tomography, we revealed the organisation of the L. aerophila envelope and propose a mechanism for how the archaellum can traverse the peptidoglycan of L. aerophila by using the Type IV pilus alignment complex proteins PilO and PilN. In addition, we identified two other cell surface appendages: (i) pilus-like structures consistent with Tad pili, and (ii) grappling hook-like structures. Structural analysis of the grappling hook by CryoEM revealed an architecture that possibly plays a role in cell-cell interactions. Together, these findings imply that the evolution of a complex, multilayered cell envelope in Chloroflexota has facilitated the functional adaptation of archaeal surface machineries, allowing these bacteria to exploit the archaellum as a simpler, more energy-efficient motility system than the bacterial flagellum.

Mammalian cells are continuously exposed to internally generated or externally applied mechanical stimuli. Mechanosensitive proteins enable cells to sense mechanical stress and induce protective mechanisms like autophagy and cytoskeletal reorientation. However, how these contribute to cellular and tissue adaptations remains largely unknown. Here, we studied the response of rat smooth muscle cells (A7r5) to uniaxial cyclic stretch. Stretching induced autophagy and adaptive actin fiber reorientation. Inhibiting autophagy using chloroquine or expressing a Bag3 (T285D-S289D) phosphosite mutant that impairs chaperone-assisted selective autophagy (CASA) delayed reorientation. Proteomic analysis revealed a depletion of cytoskeletal and focal adhesion proteins after stretching, which was attenuated by autophagy inhibition. Stretching caused a reduction in focal adhesion (FA) size, and the remodeled FAs reoriented perpendicularly to the strain direction. Concurrently, prolonged stretching activated mitochondria, and inhibiting mitochondrial ATP synthesis slowed actin reorientation, suggesting that mitochondrial activity supports the mechanoresponse. Our findings highlight the role of autophagy and mitochondria in the structural remodeling of cells upon adaptation to mechanical stress.

Mitochondrial proteostasis depends on precise N-terminal processing of imported precursor proteins. Defects in this maturation step are implicated in disease, yet the functional impact in humans remains unclear. Here we show that the intermediate cleaving peptidase ICP55, which removes a single amino acid, acts as a key stabilizer of multimeric mitochondrial protein complexes. Using proteomics and complexome profiling, we identify over 100 human ICP55 substrates and demonstrate that loss of ICP55 triggers widespread destabilization of protein assemblies, with a global shift toward smaller subcomplexes. Thus, we uncover a conserved, post-translational mechanism that safeguards mitochondrial proteostasis by regulating complex integrity through a single amino-acid cleavage, and we reveal N-terminal proteoform control as an unexpected layer of organellar homeostasis.

Plant growth-promoting bacteria can enhance plant performance under nutrient limitation, yet the underlying plant molecular responses remain incompletely resolved. We investigated growth promotion by Pseudomonas koreensis in Brachypodium distachyon under contrasting nitrogen (N) regimes using time-resolved phenotyping, elemental analysis, lipidomics and proteomics. Shoot phenotyping revealed rapid responses to N availability, whereas beneficial effects of bacterial inoculation emerged only during prolonged growth under low N. Under N limitation, inoculated plants accumulated significantly more biomass and total N than uninoculated controls, reaching levels comparable to high N plants, while no inoculation effect was observed under high N. Biomass increases were accompanied by only modest changes in tissue N concentration, indicating enhanced whole-plant N-use efficiency rather than disproportionate N enrichment. Proteomics identified N availability as the primary determinant of proteome structure, with bacterial inoculation under low N conditionally modulating selected modules towards High N states. Lipidomic profiles were largely N-driven, with only transient inoculation effects at early stages. Despite the presence of N fixation-associated genes in P. koreensis, δ15N analyses did not support substantial in planta N fixation. Together, these results support a plant-centric model in which bacterial inoculation enhances growth under N limitation by modulating plant-encoded N acquisition and metabolic organization within an N-defined framework.

Abstract Myofibrillar myopathy 6 is a rare, autosomal-dominant neuromuscular disorder caused by an amino acid exchange Pro209Leu in the co-chaperone BAG3, which disrupts muscle protein turnover and causes severe muscle weakness and shortened lifespan. We generated transgenic mice overexpressing the human mutant BAG3P209L-GFP, which rapidly develop skeletal muscle weakness unlike controls expressing BAG3WT-GFP. Here we show that mutant mice exhibit sarcomere breakdown, inflammation, protein aggregates, centralized nuclei and mitochondrial defects in their skeletal muscles, thereby reducing contraction force by ~90%. Omics profiling uncovered impaired protein synthesis, blocked autophagy, impaired mitophagy and loss of sarcomere proteins. Pathway modulation in vitro and in vivo showed autophagy dysfunction as the primary driver for the pathology, while BAG3 knockdown gene therapy markedly restored muscle function in vivo. In summary, this model recapitulates core disease features, revealing how BAG3 aggregates and loss of BAG3 function impair autophagy to drive muscle degeneration.

The MAGOH and MAGOHB paralogs are core components of the Exon Junction Complex (EJC). Previous studies have established that their simultaneous depletion is lethal, underscoring their essentiality. However, their potential redundancy and individual roles remain poorly defined. To dissect their specific functions, we generated MAGOH or MAGOHB knockout cell lines. We demonstrate that either paralog alone is sufficient to maintain core EJC functions, but their redundancy does not extend to cellular proliferation, as individual MAGOH and MAGOHB knockouts exhibit significant growth defects. Underlying this, proteomic analysis revealed distinct, paralog-specific dysregulation. MAGOH loss uniquely downregulated the mitochondrial ADP/ATP carrier SLC25A4, whereas MAGOHB loss specifically impaired PI3K-Akt signalling. This study reveals a critical non-redundancy, where MAGOH and MAGOHB buffer core EJC function but perform specialised roles in regulating mitochondrial metabolism and proliferative signalling to maintain cellular fitness.

Abstract Resistance exercise (RE) improves strength and muscle mass, with multiple benefits for human health. However, intense RE also induces acute myofibrillar damage. The molecular mechanisms that preserve, mark, degrade, and restore damaged proteins to keep skeletal muscle working under RE are incompletely understood. Based on repeated sampling of human skeletal muscle, we show here that acute, repeated and interrupted RE induce dynamic changes of the protein landscape associated with the sarcomeric cytoskeleton. These changes correlate with changes in phosphorylation indicative of adaptation and deadaptation signaling footprints. Regulation mainly affects the protein network linked to the muscle maintenance protein BAG3, which includes mechanosensory proteins, small heat shock proteins, and a lipid droplet associated protein. All network components exhibit altered phosphorylation and increased cytoskeletal association after damaging RE. Moreover, network components cooperate to recognize strained skeletal muscle structures and mediate their degradation through chaperone-assisted selective autophagy (CASA). Our study thus identifies key regulators of skeletal muscle homeostasis in humans.

Defective ion channel turnover and clearance of damaged proteins are associated with aging and neurodegeneration. The L-type Ca V 1.2 voltage-gated calcium channel mediates depolarization-induced calcium signals in heart and brain. Here, we determined the interaction surface between actin and two calcium channel subunits, Ca V β 2 and Ca V β 4 , using cross-linking mass spectrometry and protein-protein docking, and uncovered a role in replenishing conduction-defective Ca V 1.2 channels. Computational and in vitro mutagenesis identified hotspots in Ca V β that decreased the affinity for actin but not for Ca V 1.2. When coexpressed with Ca V 1.2, none of the tested actin-association-deficient Ca V β mutants altered the single-channel properties or the total number of channels at the cell surface. However, coexpression with the Ca V β 2 hotspot mutant downregulated current amplitudes, and with a concomitant reduction in the number of functionally available channels, indicating that current inhibition resulted from a build-up of conduction silent channels. Our findings established Ca V β 2 –actin interaction as a key player for clearing the plasma membrane of corrupted Ca V 1.2 proteins to ensure the maintenance of a functional pool of channels and proper calcium signal transduction. The Ca V β–actin molecular model introduces a potentially druggable protein-protein interface to intervene Ca V -mediated signaling processes.

Litorilinea aerophila , a filamentous bacterium of the phylum Chloroflexota (class Caldilineae ), exhibits unique morphological and cell envelope features that challenge traditional bacterial models. Initially described as Gram-negative, Chloroflexota are increasingly considered as monoderm, lacking a true outer membrane. In this study, we investigated the growth behaviour, cell morphology, and cell appendages of Litorilinea aerophila using light microscopy, cryo-electron tomography, and structural biology. Dry weight-based growth assays revealed a prolonged lag phase (~40 h) followed by exponential and stationary phases. Light and fluorescence microscopy revealed irregular indentations along cell filaments, accompanied by a diffuse distribution of DNA, indicating a multicellular organization. Thin-section electron microscopy confirmed septa formation, and in late growth stages, filaments became shorter with more defined indentations and membrane vesicle release. Next to the already characterised bacterial archaellum of Litorilinea aerophila , two additional types of surface appendages were identified: (i) pilus-like structures consistent with Tad pili, and (ii) grappling hook like structures. These findings contribute to the understanding of cell envelope diversity, growth, and surface structures in filamentous Chloroflexota. ### Competing Interest Statement The authors have declared no competing interest. German Reserach Foundation, 403222702-SFB 1381, CIBSS-Exc_2189-Project ID 390939984

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5´-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3' exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.

Phytocytokines are peptide signaling molecules in plant immunity. Extracellular phytocytokines and their cognate membrane-receptors have been described in various species; however, processing and release of these signals remains largely unknown. The Zea mays immune peptide 1 (Zip1) is a maize-specific phytocytokine, which is associated to the salicylic acid (SA) signaling pathway. Zip1 resides central in its precursor PROZIP1, thus C- and N-terminal cleavage is required to release bioactive Zip1 peptide. Two apoplastic maize PLCPs, CP1 and CP2, were previously shown to cleave PROZIP1. We investigated the localization and processing steps of PROZIP1 leading to Zip1 release and found that PROZIP1 undergoes processing in the endoplasmic reticulum (ER) and cytoplasm, where the N-terminal PROZIP1 is cleaved. C-terminal PROZIP1 translocates to the apoplast via an unconventional secretion pathway, likely involving exocyst-positive organelles (EXPO). The combination of antibody detection, protease cleavage assays and mass spectrometry provided evidence for a proteolytic cascade, in which intracellular processing of PROZIP1 is executed by the calcium-dependent metacaspases ZmMC9 through arginine-dependent cleavage. After secretion, C-terminal PROZIP1 is processed by apoplastic PLCPs, which release, but also degrade the Zip1 peptide. Together, these findings reveal a two-step mechanism of phytocytokine processing, translocation, activation and clearance. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, CEPLAS, EXC 2048/1project ID: 390686111, DO 1421/5-2, SFB1403 (project no. 414786233)

Proteases degrade proteins that are damaged, misfolded, or no longer needed. This enables plants to repurpose their proteomes in response to environmental cues and adapt to adverse conditions. Proteases also play key roles in plant growth and development by the selective degradation of regulatory proteins. However, there is much more to proteases than just protein degradation. They can also act in a highly site-specific manner, ensuring protein maturation after subcellular targeting, regulating protein activity and function, and releasing or modulating peptide signals. Since their activity is irreversible and potentially deleterious, proteases are tightly controlled by endogenous inhibitors and other regulatory mechanisms. The reviews and research articles in this Special Issue put a timely spotlight on the fascinating diversity, function, and regulation of proteases in plants, discuss current challenges and technical developments, and provide a perspective on their use for crop protection.

TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 to nuclear speckle condensates independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal TAF2 proximity to RNA splicing factors including specific interactions of the TAF2 IDR with SRRM2 in nuclear speckles. Deleting the IDR from TAF2 does not majorly impact global gene expression but results in changes of alternative splicing events. Further, genome-wide binding analyses suggest that the TAF2 IDR impedes TAF2 promoter association by guiding TAF2 to nuclear speckles. This study demonstrates that an IDR within the large multiprotein complex TFIID controls nuclear compartmentalization and thus links distinct molecular processes, namely transcription initiation and RNA splicing.

Protein N-termini encode essential biological information, reflecting not only the identity of the translation start site but also a range of co- and post-translational modifications (PTMs), including N-terminal acetylation, myristoylation, and proteolytic processing events. These modifications are critical for regulating protein stability, localization, and function. However, standard bottom-up proteomics workflows typically focus on internal tryptic peptides and often fail to comprehensively capture N-terminal peptides, leading to an underrepresentation of the N-terminome in global proteomic datasets. To address this gap, N-terminal peptides can be enriched by negative selection, which modifies free primary amines before proteome digestion to enable simultaneous enrichment endogenously modified and protease-generated N-terminal peptides by depletion of internal and C-terminal peptides. Here we present an updated, 2-day step-by-step protocol for High-efficiency Undecanal-based Enrichment of N-termini (HUNTER) combined with Data-Independent Acquisition (DIA) mass spectrometry for deep and reproducible N-terminome profiling. To support broad adoption, we provide pre-configured FragPipe and DIA-NN search templates optimized for N-terminomics data, as well as an open-source R/Quarto pipeline for automated downstream analysis. This includes annotation of cleavage sites and PTMs, classification of native and protease-generated neo-N-termini, and statistical analysis of differential abundance across conditions. HUNTER-DIA consistently achieves high N-terminal labeling and internal peptide depletion efficiencies, enabling sensitive detection of endogenous processing events and dynamic N-terminal PTMs across diverse sample types. This platform opens new opportunities for studying protease biology, N-terminal post-translational modifications, and their context-specific regulation in health and disease across virtually any organism.

Auxin is a crucial phytohormone that regulates plant development and facilitates dynamic responses to environmental changes through subcellular control mechanisms. PIN-LIKES (PILS) are auxin transport facilitators at the endoplasmic reticulum (ER) that mediate nuclear auxin abundance and signaling. Although the posttranslational regulation of PILS is important for acclimating growth responses, the molecular mechanisms involved remain largely unknown. This study demonstrates that components of the ER-associated degradation (ERAD) machinery regulate the proteasome-dependent degradation of functional PILS proteins under nonstressed conditions. We further reveal that both internal and external signals use the ERAD complex to differentially modulate the turnover rates of PILS proteins. Our findings uncover an additional physiological role of the ERAD complex in regulating PILS protein turnover. This finding uncovers the interplay between protein homeostasis at the ER and growth regulation, opening unexplored molecular avenues into how plants acclimate to internal and external cues.

The structure of human coagulation factor XIII (FXIII), a heterotetrameric plasma protransglutaminase that covalently cross-links preformed fi brin polymers, remains elusive until today. The heterotetrameric complex is composed of 2 catalytic FXIII-A and 2 protective FXIII-B subunits. Structural etiology underlying FXIII deficiency has so far been derived from crystallographic structures, all of which are currently available for the FXIIIA2 homodimer only. Here, we present the cryogenic electron microscopy (cryo-EM) structure of a native, human plasma-derived FXIII-A2B2 complex at 2.4 & Aring; resolution. The structure provides detailed information on FXIII subunit interacting interfaces as the 2 subunits interact strongly in plasma. The native FXIII-A2B2 complex reveals a pseudo- symmetric heterotetramer of 2 FXIII-B monomers intercalating with a symmetric FXIII-A2 dimer forming a " crown "-like assembly. The symmetry axes of the A2 and B2 homodimers are twisted relative to each other such that Sushi domain 1 interacts with the catalytic core of the A subunit, and Sushi domain 2 with the symmetry related A ' subunit, and vice versa. We also report 4 novel mutations in the F73A7 gene encoding the FXIII-A subunit from a cohort of patients with severe FXIII deficiency. Our structure reveals the etiological basis of homozygous and heterozygous pathogenic mutations and explains the conditional dominant negative effects of heterozygous mutations. This atomistic description of complex interfaces is consistent with previous biochemical data and shows a congruence between the structural biochemistry of the FXIII complex and the clinical features of FXIII deficiency.