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.

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.

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.

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).

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.

Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A.

Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.

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.

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.

Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis.

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell-specific Golgi-localized type II integral membrane glycoprotein.

ARFGAP1 is dynamically associated with lipid droplets in hepatocytes.

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation.

Assessment of internalization and endosomal signaling: studies with insulin and EGF.

Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form.

Cell biology of the endoplasmic reticulum and the Golgi apparatus through proteomics.

Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function.

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.

Golgi and related vesicle proteomics: simplify to identify.

Despite more than six decades of successful Golgi research, the fundamental question as to how biosynthetic material is transported through the secretory pathway remains unanswered. New technologies such as live cell imaging and correlative microscopy have highlighted the plastic nature of the Golgi, one that is sensitive to perturbation yet highly efficient in regaining both structure and function. Single molecule-microscopy and super resolution-microscopy further adds to this picture.

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 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.

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.

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.

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.

Cell biology through proteomics--ad astra per alia porci.

Isolated subcellular fractions have been instrumental in elucidating cell function. The use of such fractions for the identification and biochemical characterization of subcellular organelles, combined with cell- free systems, has provided key insights into the function and machineries of organelles, including those involved in vesicle transport, quality control and protein sorting. Despite their obvious utility, popular cell biology has come to regard in vitro-based approaches as inferior to in vivo-based approaches.

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.

Sorting out glycosylation enzymes in the Golgi apparatus.

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of anin vitrotransport assay by Rothman and co-workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co-workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted.

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.