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CEP peptide hormones have key roles in nitrogen-demand signalling, root nodulation, and lateral root development. We highlight recent advances in our understanding of CEP biology, from peptide biogenesis to function. Abstract Secreted peptide hormones play pivotal roles in plant growth and development. So far, CEPs (C-TERMINALLY ENCODED PEPTIDEs) have been shown to act through CEP receptors (CEPRs) to control nitrogen (N)-demand signalling, nodulation, and lateral root development. Secreted CEP peptides can enter the xylem stream to act as long-distance signals, but evidence also exists for CEPs acting in local circuits. Recently, CEP peptide species varying in sequence, length, and post-translational modifications have been identified. A more comprehensive understanding of CEP biology requires insight into the in planta function of CEP genes, CEP peptide biogenesis, the components of CEP signalling cascades and, finally, how CEP peptide length, amino-acid composition, and post-translational modifications affect biological activity. In this review, we highlight recent studies that have advanced our understanding in these key areas and discuss some future directions.

Most peptide hormones and growth factors are matured from larger inactive precursor proteins by proteolytic processing and further posttranslational modification. Whether or how posttranslational modifications contribute to peptide bioactivity is still largely unknown. We address this question here for TWS1 (Twisted Seed 1), a peptide regulator of embryonic cuticle formation in Arabidopsis thaliana. Using synthetic peptides encompassing the N- and C-terminal processing sites and the recombinant TWS1 precursor as substrates, we show that the precursor is cleaved by the subtilase SBT1.8 at both the N and the C termini of TWS1. Recognition and correct processing at the N-terminal site depended on sulfation of an adjacent tyrosine residue. Arginine 302 of SBT1.8 was found to be required for sulfotyrosine binding and for accurate processing of the TWS1 precursor. The data reveal a critical role for posttranslational modification, here tyrosine sulfation of a plant peptide hormone precursor, in mediating processing specificity and peptide maturation.

During embryonic development and adulthood, Reelin exerts several important functions in the brain including the regulation of neuronal migration, dendritic growth and branching, dendritic spine formation, synaptogenesis and synaptic plasticity. As a consequence, the Reelin signaling pathway has been associated with several human brain disorders such as lissencephaly, autism, schizophrenia, bipolar disorder, depression, mental retardation, Alzheimer’s disease and epilepsy. Several elements of the signaling pathway are known. Core components, such as the Reelin receptors very low-density lipoprotein receptor (VLDLR) and Apolipoprotein E receptor 2 (ApoER2), Src family kinases Src and Fyn, and the intracellular adaptor Disabled-1 (Dab1), are common to most but not all Reelin functions. Other downstream effectors are, on the other hand, more specific to defined tasks. Reelin is a large extracellular protein, and some aspects of the signal are regulated by its processing into smaller fragments. Rather than being inhibitory, the processing at two major sites seems to be fulfilling important physiological functions. In this review, I describe the various cellular events regulated by Reelin and attempt to explain the current knowledge on the mechanisms of action. After discussing the shared and distinct elements of the Reelin signaling pathway involved in neuronal migration, dendritic growth, spine development and synaptic plasticity, I briefly outline the data revealing the importance of Reelin in human brain disorders.

The coronavirus spike protein (S) plays a key role in the early steps of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion. In some cases, the S protein is proteolytically cleaved at the S1-S2 boundary. In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), it has been shown that virus entry requires the endosomal protease cathepsin L; however, it was also found that infection of SARS-CoV could be strongly induced by trypsin treatment. Overall, in terms of how cleavage might activate membrane fusion, proteolytic processing of the SARS-CoV S protein remains unclear. Here, we identify a proteolytic cleavage site within the SARS-CoV S2 domain (S2', R797). Mutation of R797 specifically inhibited trypsin-dependent fusion in both cell-cell fusion and pseudovirion entry assays. We also introduced a furin cleavage site at both the S2' cleavage site within S2 793-KPTKR-797 (S2'), as well as at the junction of S1 and S2. Introduction of a furin cleavage site at the S2' position allowed trypsin-independent cell-cell fusion, which was strongly increased by the presence of a second furin cleavage site at the S1-S2 position. Taken together, these data suggest a novel priming mechanism for a viral fusion protein, with a critical proteolytic cleavage event on the SARS-CoV S protein at position 797 (S2'), acting in concert with the S1-S2 cleavage site to mediate membrane fusion and virus infectivity.

Amyloid precursor protein (APP) is processed sequentially by the β-site APP cleaving enzyme and γ-secretase to generate amyloid β (Aβ) peptides, one of the hallmarks of Alzheimer’s disease. The intracellular location of Aβ production—endosomes or the trans -Golgi network (TGN)—remains uncertain. We investigated the role of different postendocytic trafficking events in Aβ ₄₀ production using an RNAi approach. Depletion of Hrs and Tsg101, acting early in the multivesicular body pathway, retained APP in early endosomes and reduced Aβ ₄₀ production. Conversely, depletion of CHMP6 and VPS4, acting late in the pathway, rerouted endosomal APP to the TGN for enhanced APP processing. We found that VPS35 (retromer)-mediated APP recycling to the TGN was required for efficient Aβ ₄₀ production. An interruption of the bidirectional trafficking of APP between the TGN and endosomes, particularly retromer-mediated retrieval of APP from early endosomes to the TGN, resulted in the accumulation of endocytosed APP in early endosomes with reduced APP processing. These data suggest that Aβ ₄₀ is generated predominantly in the TGN, relying on an endocytosed pool of APP recycled from early endosomes to the TGN.

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1β units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)₂(α2)n-2(β)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(β)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp’s antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp’s capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor’s genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

Peptide hormones are defined as small secreted polypeptide-based intercellular communication signal molecules. Such peptide hormones are encoded by nuclear genes, and often go through proteolytic processing of preproproteins and post-translational modifications. Most peptide hormones are secreted out of the cell to interact with membrane-associated receptors in neighboring cells, and subsequently activate signal transductions, leading to changes in gene expression and cellular responses. Since the discovery of the first plant peptide hormone, systemin, in tomato in 1991, putative peptide hormones have continuously been identified in different plant species, showing their importance in both short- and long-range signal transductions. The roles of peptide hormones are implicated in, but not limited to, processes such as self-incompatibility, pollination, fertilization, embryogenesis, endosperm development, stem cell regulation, plant architecture, tissue differentiation, organogenesis, dehiscence, senescence, plant-pathogen and plant-insect interactions, and stress responses. This article, collectively written by researchers in this field, aims to provide a general overview for the discoveries, functions, chemical natures, transcriptional regulations, and post-translational modifications of peptide hormones in plants. We also updated recent discoveries in receptor kinases underlying the peptide hormone sensing and down-stream signal pathways. Future prospective and challenges will also be discussed at the end of the article.

Most growth factors are synthesized as precursors and biologically active forms are generated by proteolytic cleavage of the pro-domain. However, the biological functions of pro-domains are ill-defined. New roles were recently reported for the pro-domain of brain-derived neurotrophic factor (BDNF), a well-known growth factor in the brain. Interestingly, the pro-domain of BDNF (BDNF pro-peptide) is localized at presynaptic termini, where it facilitates long-term depression (LTD) in hippocampal slices, implicating it as a novel synaptic modulator. BDNF binds its pro-peptide with high affinity in a pH-dependent manner and when bound to BDNF, the BDNF pro-peptide cannot facilitate hippocampal LTD, representing a new mechanism of regulation. The BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and levels were significantly lower in patients with major depressive disorder (MDD) than in controls. Notably, male MDD patients exhibit significantly lower levels of CSF pro-peptide than females. These findings demonstrate that the BDNF pro-peptide is a biologically important synaptic modulator and is associated with MDD, particularly in males.

Proteolytic cell surface release (‘shedding’) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

In 2019, the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) started to spread globally and caused the COVID-19 pandemic. SARS-CoV-2, like other members of the Coronaviridae , has a single-stranded, positive sense RNA genome about 30 kb in length, which is translated to generate 16 non-structural proteins (NSPs); a set of sub-genomic mRNAs encode the structural and accessory proteins. The ORF1a precursor includes NSP1-11 and is processed by virus-encoded proteases to produce the mature proteins. We recently identified a short, highly conserved motif (YCPRP) within the structural protein precursor of foot-and-mouth disease virus (FMDV), a member of the Picornaviridae. This motif is conserved among picornaviruses and is found as (W/F/Y)-x-P-R-(P/A). The motif has a major influence on the processing of the FMDV capsid precursor (P1-2A) by the viral protease 3C pro . We have now identified a similar motif (WVPRA) within the NSP2 of SARS-CoV-2. Interestingly, this motif is required for the efficient processing of the NSP1-NSP2 junction by the SARS-CoV-2 protease PL pro (NSP3) and a single amino acid substitution within the motif can abrogate cleavage of this junction. We hypothesise that this motif acts, within NSP1-NSP2, to enable this precursor to fold correctly and allow efficient processing of the NSP1/NSP2 junction.