Academic literature on the topic 'Microproteins'

MicroProteins are a class of small single-domain proteins that post-translationally regulate larger multidomain proteins from which they evolved or which they relate to. They disrupt the normal function of their targets by forming microProtein-target heterodimers through compatible protein-protein interaction (PPI) domains. Recent studies confirm the significance of microProteins in the fine-tuning of plant developmental processes such as shoot apical meristem maintenance and flowering time regulation. While there are a number of well-characterized microProteins in Arabidopsis thaliana, studies from more complex plant genomes are still missing. We have previously developed miPFinder, a software for identifying microProteins from annotated genomes. Here we present an improved version where we have updated the algorithm to increase its accuracy and speed, and used it to analyze five cereal crop genomes – wheat, rice, barley, maize and sorghum. We found 20,064 potential microProteins from a total of 258,029 proteins in these five organisms, of which approximately 2000 are high-confidence, i.e., likely to function as actual microProteins. Gene ontology analysis of these 2000 microProtein candidates revealed their roles in stress, light and growth responses, hormone signaling and transcriptional regulation. Using a recently developed rice gene co-expression database, we analyzed 347 potential rice microProteins that are also conserved in other cereal crops and found over 50 of these rice microProteins to be co-regulated with their identified interaction partners. Overall, our study reveals a rich source of biotechnologically interesting small proteins that regulate fundamental plant processes such a growth and stress response that could be utilized in crop bioengineering.

Abstract Recent work has unveiled a hidden microproteome composed by thousands of small proteins named microproteins: they are functional short proteins coded by genomic regions previously considered non-coding, and which had been completely ignored, mainly due to their small size (<100 aminoacids). To date, only a small part of the thousands of microproteins present in our cells have been characterized, and they are key players in fundamental processes such as DNA repair, mRNA splicing or cell metabolism. In cancer, they have been shown to regulate most tumor hallmarks, and present a huge potential for the clinic as diagnostic and prognostic biomarkers as well as therapeutic targets. Interestingly, their small size makes them ideal candidates to be shed in tumor-derived exosomes. PDAC-shed exosomes have been shown to prepare the pre-metastatic niche in the liver, and their presence in the bloodstream can be used as a surrogate marker of metastasis. Herein, we have mined the PDAC exosome-secreted microproteome for novel regulators of tumor progression and metastasis. Using proteogenomics in PDAC patient-derived explants, we have identified 439 microproteins secreted in exosomes by pancreatic tumors. We have selected a set of top microprotein candidates for further characterization by in silico analyses (e.g. phylogenetic conservation, predicted protein stability and localisation, mRNA expression in PDAC, etc). We have confirmed their exosome secretion in PDAC cell lines, and preliminary characterisation of these top candidates has shown that they extrinsically promote PDAC cell growth and invasion in vitro. Together, this work advances our knowledge on the underexplored field of secreted microproteins and provides pioneering evidence of their role in tumor cell communication in PDAC. It may further be a source of novel therapeutic targets and PDAC biomarkers for liquid biopsy in the clinic. Citation Format: Marion Martinez, Marta Hergueta, Pilar Ximénez de Embún, Ana Dueso, David Torrents, Teresa Macarulla, Javier Muñoz, Héctor Peinado, María Abad. Mining the secreted microproteome for novel regulators of PDAC progression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C074.

Sports genetics research began in the late 1990s and over 200 variants have been reported as athletic performance- and sports injuries-related genetic polymorphisms. Genetic polymorphisms in the α-actinin-3 (ACTN3) and angiotensin-converting enzyme (ACE) genes are well-established for athletic performance, while collagen-, inflammation-, and estrogen-related genetic polymorphisms are reported as genetic markers for sports injuries. Although the Human Genome Project was completed in the early 2000s, recent studies have discovered previously unannotated microproteins encoded in small open reading frames. Mitochondrial microproteins (also called mitochondrial-derived peptides) are encoded in the mtDNA, and ten mitochondrial microproteins, such as humanin, MOTS-c (mitochondrial ORF of the 12S rRNA type-c), SHLPs 1–6 (small humanin-like peptides 1 to 6), SHMOOSE (Small Human Mitochondrial ORF Over SErine tRNA), and Gau (gene antisense ubiquitous in mtDNAs) have been identified to date. Some of those microproteins have crucial roles in human biology by regulating mitochondrial function, and those, including those to be discovered in the future, could contribute to a better understanding of human biology. This review describes a basic concept of mitochondrial microproteins and discusses recent findings about the potential roles of mitochondrial microproteins in athletic performance as well as age-related diseases.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.

An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the extent of their prevalence is unclear. Here we present an algorithm that predicts miPs and their target TFs from a sequenced genome. The algorithm is called miP prediction program (miP3), which is implemented in Python. The software will help shed light on the prevalence, biological roles, and evolution of miPs. Moreover, miP3 can be used to predict other types of miP-like proteins that may have evolved from other functional classes such as kinases and receptors. The program is freely available and can be applied to any sequenced genome.

Abstract MicroProteins are potent post-translational regulators. In Arabidopsis (Arabidopsis thaliana), the miP1a/b microProteins delay floral transition by forming a complex with CONSTANS (CO) and the co-repressor protein TOPLESS. To better understand the function of the miP1a microProtein in floral repression, we performed a genetic suppressor screen to identify suppressors of miP1a (sum) function. One mutant, sum1, exhibited strong suppression of the miP1a-induced late-flowering phenotype. Mapping of sum1 identified another allele of the gene encoding the histone H3K4 demethylase JUMONJI14 (JMJ14), which is required for miP1a function. Plants carrying mutations in JMJ14 exhibit an early flowering phenotype that is largely dependent on CO activity, supporting an additional role for CO in the repressive complex. We further investigated whether miP1a function involves chromatin modification, performed whole-genome methylome sequencing studies with plants ectopically expressing miP1a, and identified differentially methylated regions (DMRs). Among these DMRs is the promoter of FLOWERING LOCUS T (FT), the prime target of miP1a that is ectopically methylated in a JMJ14-dependent manner. Moreover, when aberrantly expressed at the shoot apex, CO induces early flowering, but only when JMJ14 is mutated. Detailed analysis of the genetic interaction among CO, JMJ14, miP1a/b, and TPL revealed a potential role for CO as a repressor of flowering in the shoot apical meristem (SAM). Altogether, our results suggest that a repressor complex operates in the SAM, likely to maintain it in an undifferentiated state until leaf-derived florigen signals induce SAM conversion into a floral meristem.

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression ofLNJinArabidopsisresulted in stunted plants that resembled the decupleJAZ(jazD) mutant. In fact, comparing the transcriptomes of transgenicLNJoverexpressor plants andjazDrevealed a large overlap of deregulated genes, suggesting that ectopicLNJexpression altered JA signaling. Transgenic Brachypodium plants with elevatedLNJexpression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression ofLNJin barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein inArabidopsisby deleting parts of the coding sentence of theAFP2gene that encodes a NINJA-domain protein. Theseafp2-crisprmutants were also stunted in size and resembledjazD. Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

Buried seedlings undergo dramatic developmental transitions when they emerge from soil into sunlight. As central transcription factors suppressing light responses, PHYTOCHROME-INTERACTING FACTORs (PIFs) and ETHYLENE-INSENSITIVE 3 (EIN3) actively function in darkness and must be promptly repressed upon light to initiate deetiolation. Microproteins are evolutionarily conserved small single-domain proteins that act as posttranslational regulators in eukaryotes. Although hundreds to thousands of microproteins are predicted to exist in plants, their target molecules, biological roles, and mechanisms of action remain largely unknown. Here, we show that two microproteins, miP1a and miP1b (miP1a/b), are robustly stimulated in the dark-to-light transition.miP1a/bare primarily expressed in cotyledons and hypocotyl, exhibiting tissue-specific patterns similar to those ofPIFs andEIN3. We demonstrate that PIFs and EIN3 assemble functional oligomers by self-interaction, while miP1a/b directly interact with and disrupt the oligomerization of PIFs and EIN3 by forming nonfunctional protein complexes. As a result, the DNA binding capacity and transcriptional activity of PIFs and EIN3 are predominantly suppressed. These biochemical findings are further supported by genetic evidence. miP1a/b positively regulate photomorphogenic development, and constitutively expressingmiP1a/brescues the delayed apical hook unfolding and cotyledon development of plants overexpressingPIFs andEIN3. Our study reveals that microproteins provide a temporal and negative control of the master transcription factors' oligomerization to achieve timely developmental transitions upon environmental changes.

Cyclotides are a unique class of head-to-tail cyclic cysteine-knot microproteins that exhibit a wide range of biological activities ranging from insecticidal to anti-HIV. They have a unique structure in which two disulfide bridges form a ring through which a third disulfide bridge is threaded, making cyclotides exceptionally stable against thermal and proteolytic degradation. This characteristic makes them of great interest in drug discovery. MCoTI-I and MCoTI-II, which originally were extracted from Momordica cochenchinesis, are examples of macrocyclic cysteine knot microproteins that display extremely potent trypsin inhibitory activity. In this thesis, two distinct complementary approaches have been developed for the first total synthesis of MCoTI-I and MCoTI-II. In the first, termed thia zip native chemical ligation (NCL), the cyclic backbone and three disulfide bonds are formed in a one pot reaction. In the second, backbone ligation is achieved via a novel immobilised protease-mediated ligation reaction. These methodologies have been further applied to the synthesis of novel analogues of the natural MCoTI-II structure modified at a key residue in the active loop (Lys10) that have altered protease inhibition specificity together with minimizing of MCoTI-II size. These wild-type and re-engineered MCoTI cyclotides were found to be active against not only trypsin-like serine proteases; trypsin, chymotrypsin, leukocyte elastase, HCV NS3/4A, β-tryptase and matriptase, but they are also active against cysteine protease; foot-and-mouth disease virus (FMDV 3Cpro).

Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations. Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used. Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.

Au cours d’études sur la polyadénylation alternative (APA), des transcrits courts terminant dans un exon final alternatif ont été découverts, on parle de polyadénylation intronique (IPA). L’IPA est régulée par des facteurs de l’épissage (dont U1 snRNP), de polyadénylation, et d’élongation de la transcription (dont CDK12). Les isoformes IPA sont régulées par des agents génotoxiques (induisant des dommages à l’ADN), dont les rayonnements UV et la doxorubicine. Les inhibiteurs de CDK12 augmentent l’IPA dans des gènes de réparation de l’ADN et la sensibilité cellulaire à des génotoxiques. L’IPA a souvent lieu dans la région codante des gènes, générant des protéines altérées en carboxy terminal. Cependant, des transcrits IPA sont aussi générés dans les premiers introns des gènes, on parle alors de 5’IPA. Des transcrits 5’IPA sont dégradés par l’exosome nucléaire, mais certains sont abondants et ont un faible potentiel codant. Deux d’entre eux, issues des gènes ASCC3 et CDKN1A, ont des fonctions non codantes. Par ailleurs, des études montrent par Ribo-seq et spectrométrie de masse (MS) l’existence, dans des ARNm et des lncRNA, de petits cadres de lecture ouverts (sORF) codant des microprotéines (miP, protéines de moins de 100 aa) qui peuvent être fonctionnelles. Aucune miP n’a été rapportée dans des isoformes 5’IPA. Le cisplatine (CisPt) est un agent pontant de l'ADN très utilisé dans les cancers du poumon non à petites cellules (NSCLC). Mon équipe a observé par 3’-seq dans des cellules NSCLC que le CisPt augmente l’expression des isoformes IPA par rapport aux ARNm canoniques (ratio IPA:LE) dans de nombreux gènes, et que certaines isoformes IPA sont peu engagées dans les polysomes lourds et sont issues de la région en amont du site d’initiation de la traduction annoté du gène (isoformes 5’UTR-IPA). Mes objectifs étaient de déterminer le rôle de l’IPA dans la réponse cellulaire au CisPt. Je me suis intéressé premièrement au rôle des isoformes 5’UTR-IPA. Pour deux d’entre elles, issues des gènes PRKAR1B et PHF20, j’ai montré que leur déplétion par siARN augmente la survie de cellules NSCLC au CisPt. Ces deux isoformes sont engagées dans des fractions polysomiques légères. Des analyses de bases de données de Ribo-seq et de MS ont révélé l’existence de sORF dans ces deux isoformes. Par transfection de vecteurs contenant ces isoformes 5’UTR-IPA et en balisant leurs sORF, j’ai observé en ImmunoFluorescence (IF) et Western Blot que l’isoforme 5’UTR-IPA de PRKAR1B code une miP. La délétion de cette isoforme IPA endogène ou la mutation de l’ATG de sa sORF par CRISPR ont donné un phénotype similaire au siARN. Il s’agit de la première isoforme 5’UTR-IPA codant une miP (miP-5’UTR-IPA). Par croisement de nos données de 3’-seq avec des données de Ribo-seq et de MS, nous avons identifié une centaine d’isoformes miP-5’UTR-IPA potentielles induites par le CisPt. Deuxièmement, je me suis intéressé à la possibilité de sensibiliser des cellules NSCLC au CisPt en ciblant U1 snRNP par un oligonucléotide (U1-AMO) qui induit l’IPA dans de nombreux gènes. Dans plusieurs lignées cellulaires NSCLC, j’ai pu montrer une sensibilisation par U1-AMO au CisPt en termes d’inhibition de croissance cellulaire et d’induction de dommages à l’ADN (foyers ƴH2AX). Cette sensibilisation est liée à une réduction (en 3’-seq et RT-qPCR) de l'expression des ARNm canoniques dans des gènes de réparation des pontages de l'ADN (voies de Fanconi et de l’excision de nucléotides). Cependant, U1-AMO prévient les blocages de cycle cellulaire induits par le CisPt, ainsi que les effets du CisPt sur le ratio IPA:LE de nombreux gènes. Mes travaux montrent l’impact de l’IPA sur la réponse des cellules cancéreuses au CisPt, et révèlent un nouveau paradigme génétique, appelé miP-5’UTR-IPA, dans lequel des gènes produisent par IPA des transcrits courts codant des miP
During studies on alternative polyadenylation (APA), short transcripts ending in an alternative last exon were discovered, known as intronic polyadenylation (IPA). IPA is regulated by splicing factors (including U1 snRNP), polyadenylation factors and transcription elongation factors (including CDK12). IPA isoforms are regulated by genotoxic agents (inducing DNA damage), including UV radiation and doxorubicin. Conversely, CDK12 inhibitors increase both IPA in DNA repair genes and cellular sensitivity to genotoxic agents. IPA often occurs in the coding region of genes, generating carboxy-terminally altered proteins. However, IPA transcripts are also generated in the first introns of genes, known as 5'IPA. Many 5'IPA transcripts are degraded by the nuclear exosome, but some are abundant and have a low coding potential. Two of these, derived from the ASCC3 and CDKN1A genes, have non-coding functions. In addition, studies using Ribo-seq and mass spectrometry (MS) are showing the existence -in mRNAs and lncRNAs- of small open reading frames (sORF) encoding microproteins (miP, proteins of less than 100 aa) which can be functional. No miP has been reported in 5'IPA isoforms. Cisplatin (CisPt) is a DNA-crosslinking agent widely used in non-small cell lung cancer (NSCLC). My team observed, by 3'-seq in NSCLC cells, that CisPt increases the expression of IPA isoforms compared to canonical mRNAs (IPA:LE ratio) in many genes, and that some IPA isoforms are poorly associated with heavy polysomes and are derived from the region upstream of the annotated translation initiation site of the gene (5'UTR-IPA isoforms). My objectives were to determine the role of IPA in cell response to CisPt. I first looked at the role of 5'UTR-IPA isoforms. For two of them, derived from the PRKAR1B and PHF20 genes, I showed that their depletion by siRNA increased the survival of NSCLC cells to CisPt. These two isoforms are associated with light polysomal fractions. Analyses of Ribo-seq and MS databases revealed the existence of sORFs in these two isoforms. By transfecting vectors containing these 5'UTR-IPA isoforms and by tagging their sORFs, I observed by ImmunoFluorescence (IF) and Western Blot that the 5'UTR-IPA isoform of PRKAR1B encodes a miP. Deletion of this IPA isoform or mutation of the sORF ATG endogenously by CRISPR gave a phenotype similar to the siRNAs. This is the first 5'UTR-IPA isoform encoding a miP (miP-5'UTR-IPA). By cross-referencing our 3'-seq data with Ribo-seq and MS data, we identified around a hundred potential miP-5'UTR-IPA isoforms induced by CisPt. Secondly, I investigated the possibility of sensitizing NSCLC cells to CisPt by targeting U1 snRNP with an antisense oligonucleotide (U1-AMO), that induces IPA in many genes. In several NSCLC cell lines, I showed sensitization to CisPt by U1-AMO in terms of cell growth inhibition and DNA damage induction (ƴH2AX foci). This sensitization is linked to a reduced expression of the canonical mRNAs of DNA crosslinks repair pathways (Fanconi and nucleotide excision repair), as shown by 3'-seq and RT-qPCR. However, U1-AMO prevented CisPt- induced cell cycle block and the effects of CisPt on the IPA:LE ratio of many genes. My work shows the impact of IPA on the response of cancer cells to CisPt, and reveals a new genetic paradigm, called miP-5'UTR-IPA, in which genes produce short miP-encoding transcripts by IPA

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. Further, in the hematopoietic system stem cell aging is characterized by accumulation HSCs with poor self-renewal capacity and myeloid biased differentiation. Despite improved appreciation of the cell intrinsic and cell extrinsic mechanisms driving age-associated HSC functional exhaustion, no interventions have proven effective in delaying HSC aging to date. Here, we show that genetic inactivation of the Phf6 prevents age- associated HSC functional decline. Immunophenotypic and single cell transcriptomics profiling demonstrated markedly decreased accumulation of immunophenotypically-defined HSCs, reduced myeloid bias and decreased upregulation of transcriptional programs associated with stem cell aging in old hematopoietic-specific Phf6 knockout mice. Functionally, Phf6 knockout HSCs from aged mice demonstrated increased hematopoietic reconstitution capacity and preservation of lymphoid differentiation potential. Mechanistically, analysis of long-term HSCs from old Phf6 knockout mice revealed reduced levels of ongoing DNA damage and downregulation of genotoxic stress-induced transcriptional signaturesconducive of HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging, whose inactivation counters the functional deterioration of HSC activity induced with age. Microprotein encoding genes are a class of genes which encode poly-peptide gene products comprised by 100 or fewer amino acids. Until recently, many such genes had been considered of low- or no-coding potential given the technical limitations associated with identification of such small proteins. However, recently prominent examples of microprotein encoding genes have been reported with a wide variety of regulatory functions. Therefore, we hypothesized that novel microprotein genes exist within the human genome with oncogenic and tumor suppressive roles. To test this hypothesis, we developed a pipeline for identification of microproteins based on conservation of the open reading frame. Leveraging PLATE-seq to generate a high-dimensional readout in a loss-of-function screen, we then screened for microproteins with potential tumor suppressive or oncogenic function. From this, we identified a brain- specific, 65 amino-acid microprotein encoded in within LINC00617 (TUNAR) which is conserved at the protein level across vertebrates. We experimentally validated the protein-level expression of the TUNAR microprotein. In vitro and in vivo knockout and overexpression experiments demonstrate a role for TUNAR as a tumor suppressor in glioma. Specifically, we show that loss of Tunar in the mouse brain results in lower expression of Fermt1 and genes in the integrin signaling pathway. Consistently, overexpression of TUNAR in human glioblastoma multiforme cell lines significantly impeded cellular migration suggesting a role of Tunar in glioma cell dissemination. Finally, human glioma sequencing and copy number data were mined to determine the prognostic significance of the loss of TUNAR in human gliomas. These analyses demonstrated that copy number loss of TUNAR is associated with poor outcomes in lower grade gliomas and that TUNAR expression and glioma grade are strongly, negatively correlated suggesting that TUNAR likely has tumor suppressive effects in human glioma.

Cystine-knot peptides are members of the large family of Cysteine-rich proteins with a dimension typically less than 50 amino acids in their mature form, characterized by the presence of six conserved cysteine (Cys) residues linked by three disulfide bonds in a knotted arrangement (Rees & Lipscomb, 1980). Peptides containing the knot motif are widespread in various species such as fungi, insects, mollusks, and mammals, where they mainly play a defense role against microorganisms, acting as toxins, or as signals in cell defense pathways (Craik et al., 2001; Iyer & Acharya, 2011; Schwarz, 2017; Vitt et al., 2001). In plants, cystine-knot peptides (also referred to as cysteine-knot miniproteins) are often involved in resistance to pathogens with the function of protease inhibitors, namely metallocarboxypeptidases and serine proteases (Daly & Craik, 2011; Molesini et al., 2017). A class of cystine-knot protease inhibitors specific to the Solanaceae family was described for the first time in the 1980s (Hass & Hermodson, 1981a; Rees & Lipscomb, 1980). This class includes the tomato cystine-knot miniproteins 1 and 2 (TCMP-1 and TCMP-2), of 37 and 44 amino acids, respectively. TCMP-1 and TCMP-2 display a sequential expression pattern, which is highly modulated during flower and fruit development. TCMP-1 is expressed at a very high level in flower buds before anthesis, then its expression decreases rapidly after anthesis and increases again during fruit development (Cavallini et al., 2011). TCMP-1 mRNA level is very low in leaves, although its expression is induced by wounding and elicitors of responses to biotic stress (Díez-Díaz et al., 2004; Martineau et al., 1991). The expression of TCMP-2 is low in flower buds before anthesis, and gradually increases after fertilization, reaching a maximum in green and ripe fruits, whereas it is apparently absent in leaves, roots, and stems (Cavallini et al., 2011; Pear et al., 1989; Treggiari et al., 2015). Indeed, the TCMP-2 promoter (also named 2A11; X13743; [Pear et al., 1989]) has been successfully used to improve qualitative trait in tomato fruit (Davuluri et al., 2005). Although the biological activity of metallocarboxypeptidase inhibitors supports a role for Solanaceous cystine-knot proteins in plant defense, it has recently been demonstrated that tomato TCMPs are implicated in fruit development (Molesini et al., 2018). In the paper by Molesini et al. (2018), tomato plants transformed with a chimeric gene containing the entire TCMP-1 coding region under the control of the TCMP-2 promoter (pTCMP-2::TCMP-1) showed a marked increase in the expression of TCMP-2 before anthesis, associated with anticipated fruit production. This evidence suggests that TCMPs are regulators of fruiting time and that the maintenance of a correct TCMP-1/TCMP-2 ratio is required for proper initial fruit growth. The mode of action of TCMPs remains largely unexplored also due to the absence of homologous genes in other model species, including Arabidopsis thaliana. In several cases, Cysteine-rich peptides act as signaling molecules in plant development, by interfering with receptors or modifying the activity of multimeric complexes (de Coninck & de Smet, 2016; Tavormina et al., 2015). The general aim of this thesis was a further investigation of the functional role played by TCMP-2 during the first phases of reproductive development. Specific aims were: 1) phenotypic and molecular analyses of pTCMP-2::TCMP-1 and 35S::TCMP-2 plants, during the transition from vegetative growth to reproductive development; 2) identification of TCMP-2 interacting partners by Yeast Two-Hybrid (Y2H) screening using a tomato cDNA library; 3) functional study of one of these interactors (i.e., a B-BOX motif-containing protein) in both Arabidopsis and tomato. A detailed analysis of pTCMP-2::TCMP-1 plants during the transition from the vegetative to the reproductive stage showed an anticipated termination of the sympodial units linked with an induced expression of the florigen gene SINGLE FLOWER TRUSS (SFT), which is the main inducer of flowering. Moreover, MicroTom plants over-expressing TCMP-2 under the control of the CaMV35S constitutive promoter exhibited a reduction of the primary shoot length, very often accompanied by a decreased number of leaves before the first inflorescence, which are indicators of early flowering. The Y2H screen permitted the identification of 47 potential interactors of TCMP-2. Among them, we focused on the B-Box domain-containing protein 16 (SlBBX16). The interaction between TCMP-2 and SlBBX16 was validated in vivo in plant cells by bimolecular fluorescence complementation (BIFC) analysis. We demonstrated that TCMP-2 is also able to interact with SlBBX17, which is the closest tomato homolog of SlBBX16, and with the SlBBX16 Arabidopsis homolog, AtBBX31. A recent study showed the involvement of AtBBX30 and AtBBX31 microproteins (also referred to as miP1b and miP1a) in a multiprotein complex which regulates flowering time in Arabidopsis (Graeff et al., 2016). These proteins interact with the flowering regulator CONSTANS (AtBBX1) and additionally engage in a larger protein complex involving the co-repressor protein TOPLESS through a specific amino acid motif (PFVFL). These interactions suppress the CO-mediated induction of FT expression, causing the severe late flowering phenotype observed in plants over-expressing AtBBX30/31. The implication of AtBBX30/31 in flowering control may indicate that the TCMP-2-SlBBX16/SlBBX17 interaction could play a role in the regulation of flowering. To test this hypothesis, we ectopically overexpressed TCMP-2, SlBBX16 and SlBBX17 in Arabidopsis. The overexpression of TCMP-2 led to an anticipated flowering phenotype linked to an increased FT mRNA level, whereas the overexpression of SlBBX16 and SlBBX17 in Arabidopsis WT and AtBBX30/31 KO mutant resulted in a slight delay in flowering time, suggesting that tomato BBXs were unable to fully phenocopy AtBBX30/31 overexpression. One of the possible reasons for the weak phenotype displayed by A. thaliana over-expressing SlBBX16 and SlBBX17 could be attributed to their inability to interact with AtCO and AtTPL, since the interaction between AtBBX30/31 with AtCO and AtTPL is due to the PFVLF motif (Graeff et al., 2016), which is absent in SlBBX16 and SlBBX17. Indeed, the Y2H experiments revealed no interaction between the tomato BBXs and AtCO and AtTPL, even when using a mutated version of SlBBXs containing the PFVLF motif. The functional study in tomato was conducted on SlBBX17, which presents a peculiar expression pattern in the floral organs. MicroTom plants over-expressing SlBBX17 showed a number of leaves at the first inflorescence similar to that of WT plants, but a delay in the time to reach the flower anthesis stage and a reduced number of ripe fruits. To investigate whether in tomato TCMP-2 and SlBBX17 may participate in a multiprotein complex similar to that described in Arabidopsis (AtBBX30/31-CO-TPL), we carried out ad hoc Y2H analyses to test the interaction between TCMP-2 and SlCO1 and SlTPL1 and the interaction between SlBBX17 and SlCO1 and SlTPL1. Our data indicate that neither TCMP-2 nor SlBBX17 can directly bind to SlCO1 and SlTPL1. Further investigation of the role in flowering and fruiting of SlBBX16, the homologue of SlBBX17, may provide additional insight into the function of BBXs microproteins in tomato.

Abstract Beta 2-microglobulin ([32m) derived amyloid is found in patients with impaired renal function either as urinary concretions or as systemic, mostly osteoarticular, deposits. Such amyloid is rarely seen in the predialysis phase (Linke et al. 1986; Moriniere et al. 1991), but is a very common complication after institution of dialysis (Campistol et al. 1990; Ritz and Bommer 1988). Generation of amyloid is dependent on increased plasma [32m concentrations. The increase of plasma [32m concentration is mainly due to diminished renal excretion of the 11.8 kDa microprotein which is normally eliminated via glomerular filtration (Vincent and Revillard 1980). The high plasma [32m concentrations in dialysed patients may not simply be the result of renal retention.