Paula D. Duek

Rhythmic growth explained by coincidence between internal and external cues

Most organisms use circadian oscillators to coordinate physiological and developmental processes such as growth with predictable daily environmental changes like sunrise and sunset. The importance of such coordination is highlighted by studies showing that circadian dysfunction causes reduced fitness in bacteria and plants, as well as sleep and psychological disorders in humans. Plant cell growth requires energy and water—factors that oscillate owing to diurnal environmental changes. Indeed, two important factors controlling stem growth are the internal circadian oscillator and external light levels. However, most circadian studies have been performed in constant conditions, precluding mechanistic study of interactions between the clock and diurnal variation in the environment. Studies of stem elongation in diurnal conditions have revealed complex growth patterns, but no mechanism has been described. Here we show that the growth phase of Arabidopsis seedlings in diurnal light conditions is shifted 8–12 h relative to plants in continuous light, and we describe a mechanism underlying this environmental response. We find that the clock regulates transcript levels of two basic helix–loop–helix genes, phytochrome-interacting factor 4 (PIF4) and PIF5, whereas light regulates their protein abundance. These genes function as positive growth regulators; the coincidence of high transcript levels (by the clock) and protein accumulation (in the dark) allows them to promote plant growth at the end of the night. Thus, these two genes integrate clock and light signalling, and their coordinated regulation explains the observed diurnal growth rhythms. This interaction may serve as a paradigm for understanding how endogenous and environmental signals cooperate to control other processes.

The degradation of HFR1, a putative bHLH class transcription factor involved in light signaling, is regulated by phosphorylation and requires COP1

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorphogenesis. The ubiquitin E3 ligase COP1 (constitutively photomorphogenic 1) regulates the stability of several light-signaling components. HFR1 (long hypocotyl in far-red light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes. HFR1 is closely related to PIF1, PIF3, and PIF4 (phytochrome interacting factor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling , as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response.

neXtProt: a knowledge platform for human proteins

neXtProt (http://www.nextprot.org/) is a new human protein-centric knowledge platform. Developed at the Swiss Institute of Bioinformatics (SIB), it aims to help researchers answer questions relevant to human proteins. To achieve this goal, neXtProt is built on a corpus containing both curated knowledge originating from the UniProtKB/Swiss-Prot knowledgebase and carefully selected and filtered high-throughput data pertinent to human proteins. This article presents an overview of the database and the data integration process. We also lay out the key future directions of neXtProt that we consider the necessary steps to make neXtProt the one-stop-shop for all research projects focusing on human proteins.

Annotating single amino acid polymorphisms in the UniProt/Swiss-Prot knowledgebase.

UniProtKB/Swiss‐Prot (http://beta.uniprot.org/uniprot; last accessed: 19 October 2007) is a manually curated knowledgebase providing information on protein sequences and functional annotation. It is part of the Universal Protein Resource (UniProt). The knowledgebase currently records a total of 32,282 single amino acid polymorphisms (SAPs) touching 6,086 human proteins (Release 53.2, 26 June 2007). Nearly all SAPs are derived from literature reports using strict inclusion criteria. For each SAP, the knowledgebase provides, apart from the position of the mutation and the resulting change in amino acid, information on the effects of SAPs on protein structure and function, as well as their potential involvement in diseases. Presently, there are 16,043 disease‐related SAPs, 14,266 polymorphisms, and 1,973 unclassified variants recorded in UniProtKB/Swiss‐Prot. Relevant information on SAPs can be found in various sections of a UniProtKB/Swiss‐Prot entry. In addition to these, cross‐references to human disease databases as well as other gene‐specific databases, are being added regularly. In 2003, the Swiss‐Prot variant pages were created to provide a concise view of the information related to the SAPs recorded in the knowledgebase. When compared to the information on missense variants listed in other mutation databases, UniProtKB/Swiss‐Prot further records information on direct protein sequencing and characterization including posttranslational modifications (PTMs). The direct links to the Online Mendelian Inheritance in Man (OMIM) database entries further enhance the integration of phenotype information with data at protein level. In this regard, SAP information in UniProtKB/Swiss‐Prot complements nicely those existing in genomic and phenotypic databases, and is valuable for the understanding of SAPs and diseases. Hum Mutat 29(3), 361–366, 2008.

The neXtProt knowledgebase on human proteins: current status.

neXtProt (http://www.nextprot.org) is a human protein-centric knowledgebase developed at the SIB Swiss Institute of Bioinformatics. Focused solely on human proteins, neXtProt aims to provide a state of the art resource for the representation of human biology by capturing a wide range of data, precise annotations, fully traceable data provenance and a web interface which enables researchers to find and view information in a comprehensive manner. Since the introductory neXtProt publication, significant advances have been made on three main aspects: the representation of proteomics data, an extended representation of human variants and the development of an advanced search capability built around semantic technologies. These changes are presented in the current neXtProt update.

The neXtProt knowledgebase on human proteins: 2017 update.

The neXtProt human protein knowledgebase (https://www.nextprot.org) continues to add new content and tools, with a focus on proteomics and genetic variation data. neXtProt now has proteomics data for over 85% of the human proteins, as well as new tools tailored to the proteomics community. Moreover, the neXtProt release 2016-08-25 includes over 8000 phenotypic observations for over 4000 variations in a number of genes involved in hereditary cancers and channelopathies. These changes are presented in the current neXtProt update. All of the neXtProt data are available via our user interface and FTP site. We also provide an API access and a SPARQL endpoint for more technical applications.

Functional Identification of APIP as Human mtnB, a Key Enzyme in the Methionine Salvage Pathway

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.

Systems Proteomics View of the Endogenous Human Claudin Protein Family

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein-protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation.

Exploring the Uncharacterized Human Proteome Using neXtProt

20,230 protein-coding genes have been predicted from the analysis of the human genome (neXtProt release 2018-01-17), and about 10% of them are still lacking functional annotation, either predicted by bioinformatics tools or captured from experimental reports. A systematic exploration of the available literature on uncharacterized human genes/proteins led to proposal of functional annotations for 113 proteins and to consolidation of a list of 1,862 uncharacterized human proteins. The advanced search functionality of neXtProt was used extensively in order to examine the landscape of the uncharacterized human proteome in terms of subcellular locations, protein-protein interactions, tissue expression, association with diseases, and 3D structure. Finally, a deep data mining in various publicly available resources allowed building functional hypotheses for 26 uncharacterized human proteins validated at protein level (uPE1). These hypotheses cover the fields of cilia biology, male reproduction, metabolism, nervous system, immunity, inflammation, RNA metabolism, and chromatin biology. They will require experimental validation before they can be considered for annotation. Despite technological progresses, the pace of human protein characterization studies is still slow. It could be accelerated by a better integration of existing knowledge resources and by initiating large collaborative projects involving specialists of different biology fields. We hope that our analysis will contribute to set up the ground for such collaborative approaches and will be exploited by the HUPO Human Proteome Project teams committed to characterize uPE1 proteins.

PepPSy: a web server to prioritize gene products in experimental and biocuration workflows

Among the 20 000 human gene products predicted from genome annotation, about 3000 still lack validation at protein level. We developed PepPSy, a user-friendly gene expression-based prioritization system, to help investigators to determine in which human tissues they should look for an unseen protein. PepPSy can also be used by biocurators to revisit the annotation of specific categories of proteins based on the ‘omics’ data housed by the system. In this study, it was used to prioritize 21 dubious protein-coding genes among the 616 annotated in neXtProt for reannotation. PepPSy is freely available at http://peppsy.genouest.org. Database URL: http://peppsy.genouest.org.