Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology.

Proteomics Impact on Cell Biology to Resolve Cell Structure and Function.

The acceleration of advances in proteomics has enabled integration with imaging at the EM and light microscopy levels, cryo-EM of protein structures, and artificial intelligence with proteins comprehensively and accurately resolved for cell structures at nanometer to subnanometer resolution. Proteomics continues to outpace experimentally based structural imaging, but their ultimate integration is a path toward the goal of a compendium of all proteins to understand mechanistically cell structure and function.

mTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1.

The mechanisms that link environmental and intracellular stimuli to mitochondrial functions, including fission/fusion, ATP production, metabolite biogenesis, and apoptosis, are not well understood. Here, we demonstrate that the nutrient-sensing mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates translation of mitochondrial fission process 1 (MTFP1) to control mitochondrial fission and apoptosis. Expression of MTFP1 is coupled to pro-fission phosphorylation and mitochondrial recruitment of the fission GTPase dynamin-related protein 1 (DRP1). Potent active-site mTOR inhibitors engender mitochondrial hyperfusion due to the diminished translation of MTFP1, which is mediated by translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Uncoupling MTFP1 levels from the mTORC1/4E-BP pathway upon mTOR inhibition blocks the hyperfusion response and leads to apoptosis by converting mTOR inhibitor action from cytostatic to cytotoxic. These data provide direct evidence for cell survival upon mTOR inhibition through mitochondrial hyperfusion employing MTFP1 as a critical effector of mTORC1 to govern cell fate decisions.

Kathryn Howell (1939-2020) "The Secretory Pathway was Imprinted in her Heart".

On April 10, 2020, a treasured cell biologist and ardent champion of the Golgi complex passed away. This has caused deep sadness, and we seek to commemorate her remarkable scientific contributions, her warm and generous personality, and her endearing sense of humor.

Proteomics Identifies Golgi phosphoprotein 3 (GOLPH3) with A Link Between Golgi Structure, Cancer, DNA Damage and Protection from Cell Death.

GOLPH3 is the first example of a Golgi resident oncogene protein. It was independently identified in multiple screens; first in proteomic-based screens as a resident protein of the Golgi apparatus, and second as an oncogene product in a screen for genes amplified in cancer. A third screen uncovered the association of GOLPH3 with the Golgi resident phospholipid, phosphatidyl inositol 4 phosphate (PI4P) to maintain the characteristic ribbon structure of the Golgi apparatus favoring vesicular transport of secretory proteins.

PCSK9 deficiency unmasks a sex- and tissue-specific subcellular distribution of the LDL and VLDL receptors in mice.

Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17ß-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.

Organellar proteomics to create the cell map.

The elucidation of a complete, accurate, and permanent representation of the proteome of the mammalian cell may be achievable piecemeal by an organellar based approach. The small volume of organelles assures high protein concentrations. Providing isolated organelles are homogenous, this assures reliable protein characterization within the sensitivity and dynamic range limits of current mass spec based analysis. The stochastic aspect of peptide selection by tandem mass spectrometry for sequence determination by fragmentation is dealt with by multiple biological replicates as well as by prior protein separation on 1-D gels. Applications of this methodology to isolated synaptic vesicles, clathrin coated vesicles, endosomes, phagosomes, endoplasmic reticulum, and Golgi apparatus, as well as Golgi-derived COPI vesicles, have led to mechanistic insight into the identity and function of these organelles.

Mass spectrometry in high-throughput proteomics: ready for the big time.

Mass spectrometry has evolved and matured to a level where it is able to assess the complexity of the human proteome. We discuss some of the expected challenges ahead and promising strategies for success.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

The human proteome project: Current state and future direction.

After successful completion of the Human Genome Project (HGP), HUPO has recently officially launched a global Human Proteome Project (HPP) which is designed to map the entire human protein set. Given the presence of about 30% undisclosed proteins out of 20,300 protein gene products, a systematic global effort is necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP groups employ the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge base. The HPP participants will take advantage of the output and cross-analyses from the ongoing HUPO initiatives and a chromosome-based protein mapping strategy, termed C-HPP with many national teams currently engaged. In addition, numerous biologically-driven projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents and tools for protein studies and analyses, and a stronger basis for personalized medicine. HUPO urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

N-glycosylation mediated folding and quality control in serine proteases of the hepsin family.

N-linked glycans are specifically attached to asparagine residues in a N-X-S/T motif of secretory pathway glycoproteins. N-glycosylation of newly synthesized glycoproteins directs their folding via the lectin chaperones calnexin and calreticulin that are associated with protein-folding enzymes and glycosidases of the endoplasmic reticulum (ER). Misfolded glycoproteins are retained in the ER by the same lectin chaperones. The work by Sun et al. (FEBS J 2023, 10.1111/febs.16757) in this issue focusses on hepsin, a serine protease on the surface of liver and other organs. The authors deduce that spatial positioning of N-glycans on one side of a conserved domain of hepsin, known as the scavenger receptor-rich cysteine domain, regulates calnexin selection for hepsin maturation and transport through the secretory pathway. If N-glycosylation is elsewhere on hepsin, then it is misfolded and has a prolonged accumulation with calnexin and BiP. This association coincides with the engagement of stress response pathways that sense glycoprotein misfolding. The topological considerations of N-glycosylation dissected by Sun et al. may help unravel how key sites of N-glycosylation sites required for protein folding and transport have evolved to select the lectin chaperone calnexin pathway for folding and quality control.

Protein quality control in the ER: the recognition of misfolded proteins.

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.

Calnexin phosphorylation: linking cytoplasmic signalling to endoplasmic reticulum lumenal functions.

Calnexin is an abundant integral membrane phosphoprotein of the endoplasmic reticulum (ER) of eukaryotic cells. The role of the luminal domain as an N-glycoprotein specific lectin has been well-established. Cytosolic C-terminal domain phosphorylation of calnexin has recently been elucidated in glycoprotein folding and quality control. Signalling of the presence of unfolded proteins from the lumen of the ER is mediated by the three ER membrane sensor proteins Ire1, ATF6 and PERK. The observation that the C-terminus of calnexin is differentially phosphorylated when glycoproteins are misfolded initiated our search for functional roles of calnexin phosphorylation. Recent studies have defined a role for phosphorylation at a proline-directed kinase site (Ser563) in ER protein quality control, while phosphorylation at a casein kinase 2 site (Ser534, Ser544) may be linked to transport functions. There are also four other abundant integral membrane phosphoproteins in the ER, and these may be components of other signalling pathways that link and coordinate other ER functions with the rest of the cell.

Standard guidelines for the chromosome-centric human proteome project.

The objective of the international Chromosome-Centric Human Proteome Project (C-HPP) is to map and annotate all proteins encoded by the genes on each human chromosome. The C-HPP consortium was established to organize a collaborative network among the research teams responsible for protein mapping of individual chromosomes and to identify compelling biological and genetic mechanisms influencing colocated genes and their protein products. The C-HPP aims to foster the development of proteome analysis and integration of the findings from related molecular -omics technology platforms through collaborations among universities, industries, and private research groups. The C-HPP consortium leadership has elicited broad input for standard guidelines to manage these international efforts more efficiently by mobilizing existing resources and collaborative networks. The C-HPP guidelines set out the collaborative consensus of the C-HPP teams, introduce topics associated with experimental approaches, data production, quality control, treatment, and transparency of data, governance of the consortium, and collaborative benefits. A companion approach for the Biology and Disease-Driven HPP (B/D-HPP) component of the Human Proteome Project is currently being organized, building upon the Human Proteome Organization's organ-based and biofluid-based initiatives (www.hupo.org/research). The common application of these guidelines in the participating laboratories is expected to facilitate the goal of a comprehensive analysis of the human proteome.

A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.

We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography-mass spectrometry-based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry-based proteomics.

Brain-specific proteins decline in the cerebrospinal fluid of humans with Huntington disease.

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

Biogenesis of lipid droplets--how cells get fatter.

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.

Calnexin phosphorylation attenuates the release of partially misfolded alpha1-antitrypsin to the secretory pathway.

Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser(563) affecting calnexin association with the translocon. Here we find an additional function for calnexin phosphorylation at Ser(563) in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory alpha1-antitrypsin. Under these conditions the phosphorylation of calnexin is enhanced at Ser(563). Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of alpha1-antitrypsin secretion comparable with that achieved by inhibiting alpha-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of calnexin is linked to the efficiency of secretion of a cargo glycoprotein.

Organelle proteomics: looking at less to see more.

The recent finding that the human genome comprises between 21000 and 39000 genes, a number much lower than expected, has in no way simplified the complexity associated with the understanding of how cells perform their functions. Elucidation of the molecular mechanisms underlying cell functions will require a global knowledge of the expressed proteins, including splice variant products, their post-translational modifications, their subcellular localizations and their assembly into molecular machines as deduced from protein-protein interactions, at any given time during the life of the cell or under any cellular conditions. Current and expected advances in mass spectrometry and bioinformatics might help the realization of these goals in a shorter time than is currently predicted.