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Background: Bipolar disorder (BD) is a multifactorial psychiatric illness affecting ∼1% of the global adult population. Lithium (Li), is the most effective mood stabilizer for BD but works only for a subset of patients and its mechanism of action remains largely elusive., Methods: In the present study, we used iPSC-derived neurons from patients with BD who are responsive (LR) or not (LNR) to lithium. Combined electrophysiology, calcium imaging, biochemistry, transcriptomics, and phosphoproteomics were employed to provide mechanistic insights into neuronal hyperactivity in BD, investigate Li's mode of action, and identify alternative treatment strategies., Findings: We show a selective rescue of the neuronal hyperactivity phenotype by Li in LR neurons, correlated with changes to Na + conductance. Whole transcriptome sequencing in BD neurons revealed altered gene expression pathways related to glutamate transmission, alterations in cell signalling and ion transport/channel activity. We found altered Akt signalling as a potential therapeutic effect of Li in LR neurons from patients with BD, and that Akt activation mimics Li effect in LR neurons. Furthermore, the increased neural network activity observed in both LR & LNR neurons from patients with BD were reversed by AMP-activated protein kinase (AMPK) activation., Interpretation: These results suggest potential for new treatment strategies in BD, such as Akt activators in LR cases, and the use of AMPK activators for LNR patients with BD., Funding: Supported by funding from ERA PerMed, Bell Brain Canada Mental Research Program and Brain & Behavior Research Foundation., Competing Interests: Declaration of interests All the authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Metastasis is the principal cause of cancer treatment failure and an area of dire diagnostic needs. Colorectal cancer metastases to the liver (CRCLMs) are predominantly classified into desmoplastic and replacement based on their histological growth patterns (HGPs). Desmoplastic responds well to current treatments, while replacement HGP has a poor prognosis with low overall survival rates., Methods: We hypothesised that complex cellular response underlying HGPs may be reflected in the proteome of circulating extracellular vesicles (EVs). EV proteomics data was generated through LC-MS/MS and analysed with Maxquant and Perseus. To validate the S100A9 signature, ELISA was performed, and IHC and IF were conducted on tissue for marker detection and colocalization study., Results: Plasma EV proteome signature distinguished desmoplastic from the replacement in patients with 22 differentially expressed proteins, including immune related markers. Unsupervised PCA analysis revealed clear separation of the two lesions. The marker with the highest confidence level to stratify the two HGPs was S100A9, which was traced in CRCLM lesions and found to colocalize with macrophages and neutrophils. EV-associated S100A9 in plasma may reflect the innate immunity status of metastatic lesions and their differential therapeutic responses., Conclusion: Plasma EV-derived S100A9 could be useful in personalising therapy in patients with CRCLM., (© 2023. The Author(s).)

The physiological functions of the rhomboid-related protein 4 (RHBDL4) are emerging, but their molecular details remain unclear. Because increased expression of RHBDL4 has been clinically linked to poorer outcomes in cancer patients, this association urgently demands a better understanding of RHBDL4. To elucidate the molecular interactions and pathways that RHBDL4 may be involved in, we conducted proximity-dependent biotin identification (BioID) assays. Our analyses corroborated several of the expected protein interactors such as the transitional endoplasmic reticulum (ER) ATPase VCP/p97 (TERA), but they also described novel putative interactors including IRS4, PGAM5, and GORS2. Using proximity-ligation assays, we validated VCP/p97, COPB, and VRK2 as proteins that are in proximity to RHBDL4. Overall, our results support the emerging functions of RHBDL4 in ER quality control and also point toward putative RHBDL4 functions in protein membrane insertion and membrane organization and trafficking.

Functional oncogenic links between ErbB2 and ERRα in HER2+ breast cancer patients support a therapeutic benefit of co-targeted therapies. However, ErbB2 and ERRα also play key roles in heart physiology, and this approach could pose a potential liability to cardiovascular health. Herein, using integrated phosphoproteomic, transcriptomic and metabolic profiling, we uncovered molecular mechanisms associated with the adverse remodeling of cardiac functions in mice with combined attenuation of ErbB2 and ERRα activity. Genetic disruption of both effectors results in profound effects on cardiomyocyte architecture, inflammatory response and metabolism, the latter leading to a decrease in fatty acyl-carnitine species further increasing the reliance on glucose as a metabolic fuel, a hallmark of failing hearts. Furthermore, integrated omics signatures of ERRα loss-of-function and doxorubicin treatment exhibit common features of chemotherapeutic cardiotoxicity. These findings thus reveal potential cardiovascular risks in discrete combination therapies in the treatment of breast and other cancers., (© 2022. The Author(s).)

Increased macroautophagy/autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK (AMP-activated protein kinase) activation and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) inhibition to maintain energy homeostasis and cell survival. TFEB (transcription factor E3) and TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by MTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB and TFE3 transcriptional activity upon nutrient starvation, FLCN (folliculin) depletion and pharmacological manipulation of MTORC1 or AMPK. Collectively, we show that MTORC1 specifically controls TFEB and TFE3 cytosolic retention, whereas AMPK is essential for TFEB and TFE3 transcriptional activity. This dual and opposing regulation of TFEB and TFE3 by MTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1 (unc-51 like autophagy activating kinase 1). Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466, 467 and 469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB and TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance. Abbreviations: ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; AMPKi: AMPK inhibitor, SBI-0206965; CA: constitutively active; CARM1: coactivator-associated arginine methyltransferase 1; CFP: cyan fluorescent protein; CLEAR: coordinated lysosomal expression and regulation; DKO: double knock-out; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; EBSS: Earle's balanced salt solution; FLCN: folliculin; GFP: green fluorescent protein; GST: glutathione S-transferases; HD: Huntington disease; HTT: huntingtin; KO: knock-out; LAMP1: lysosomal associated membrane protein 1; MEF: mouse embryonic fibroblasts; MITF: melanocyte inducing transcription factor; MTORC1: MTOR complex 1; PolyQ: polyglutamine; RPS6: ribosomal protein S6; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TCL: total cell lysates; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TKO: triple knock-out; ULK1: unc-51 like autophagy activating kinase 1.

Events at a receptor ectodomain affect the intracellular domain conformation, activating signal transduction (out-to-in conformational effects). We investigated the reverse direction (in-to-out) where the intracellular domain may impact on ectodomain conformation. The primary sequences of naturally occurring TrkC receptor isoforms (TrkC-FL and TrkC.T1) only differ at the intracellular domain. However, owing to their differential association with Protein Disulfide Isomerase the isoforms have different disulfide bonding and conformations at the ectodomain. Conformations were exploited to develop artificial ligands, mAbs, and small molecules, with isoform-specific binding and biased activation. Consistent, the physiological ligands NT-3 and PTP-sigma bind both isoforms, but NT-3 activates all signaling pathways, whereas PTP-sigma activates biased signals. Our data support an "in-to-out" model controlling receptor ectodomain conformation, a strategy that enables heterogeneity in receptors, ligands, and bioactivity. These concepts may be extended to the many wild-type or oncogenic receptors with known isoforms., Competing Interests: Authors have patent holdings and applications for ligands and bioactivities that are related to the subject matter of the contribution (F.B. and H.U.S. inventors)., (© 2020 The Authors.)

The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Purpose: The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood., Experimental Approach: CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR., Key Results: The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator., Conclusions and Implications: Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770., (© 2015 The British Pharmacological Society.)

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low-folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr(+/+) mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr(+/-) animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS-related proteins, the protein synthetic machinery, and fatty acid-related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.

Cotranslational translocation of polypeptides into the ER is controlled by the dynamic interaction of ribosome and translocon components. Analysis of the steps involved in this process by high resolution techniques such as gel electrophoresis is precluded by the high molecular masses of these complexes. We show, here, that modifications to standard native electrophoresis protocols can overcome these problems and lead to an increase in mass range and resolution. Using the modified technique, we show that ER ribosome anchored membrane protein (RAMP) complexes resolve into 3 stable and semistable complexes which range in size between 4 and 8 MDa and are sensitive to relevant concentrations of divalent metals. We demonstrate the molecular composition of the complexes and identify a number of modular components that differentiate them. The components that are common to all three RAMP complexes include the OST translocon subcomplex, Glucosidase I and microtubule tethering protein CLIMP63. The two larger complexes further include the kinesin motor binding protein p180 and Sec61, and the largest complex includes the TRAP translocon component and apoptotic regulator BAP31. On the lumenal side, the BiP cochaperone ERdj3 resides with the three RAMP complexes. Our observations may hint at how subcompartmentalization is achieved in the ER membrane continuum.

Rabs and Arfs/Arls are Ras-related small GTPases of particular relevance to membrane trafficking. It is thought that these proteins regulate specific pathways through interactions with coat, motor, tether and SNARE proteins. We screened a comprehensive list of Arf/Arl/Rab proteins, previously identified on purified Golgi membranes by a proteomics approach (37 in total), for Golgi or intra-Golgi localization, dominant-negative and overexpression phenotypes. Further analysis of two of these proteins, Rab18 and Rab43, strongly indicated roles in ER-Golgi trafficking. Rab43-T32N redistributed Golgi elements to ER exit sites without blocking trafficking of the secretory marker VSVG-GFP from ER to cell surface. Wild-type Rab43 redistributes the p150(Glued) subunit of dynactin, consistent with a specific role in regulating association of pre-Golgi intermediates with microtubules. Overexpression of wild-type GFP-Rab18 or incubation with any of three siRNAs directed against Rab18 severely disrupts the Golgi complex and reduces secretion of VSVG. Rab18 mutants specifically enhance retrograde Golgi-ER transport of the COPI-independent cargo beta-1,4-galactosyltransferase (Galtase)-YFP but not the COPI-dependent cargo p58-YFP from the Golgi to ER in a photobleach assay. Rab18-S22N also potentiated brefeldin-A-induced ER-Golgi fusion. This study is the first comprehensive application of large-scale proteomics to the cell biology of small GTPases of the secretory pathway.

Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90-preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone-preprotein complexes.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

Human 2-D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi-bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2-D PAGE reference maps (non-cultured keratinocytes, non-cultured transitional cell carcinomas, MRC-5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2-D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss-Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on-line information is updated regularly.

The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two-dimensional (2-D) gel electrophoresis in combination with computer-aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To date, comprehensive 2-D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http:@biobase.dk/cgi-bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2-D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post-translational trimmings and modifications of the proteins. The method, in combination with 2-D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.

The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro. The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA. The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA. Thus, single KH domains are discrete and independent nucleic-acid-binding units. Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been identified (protein name, organelle components, etc.) using a procedure or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies, (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry, (v)vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather--through a systematic study of ekeratinocytes--qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

We have revealed and characterised two nucleic-acid-binding proteins, termed PCBP-1 (M(r) 37,525, pI 7.07) and PCBP-2 (M(r) 38,579, pI 6.76), that together with heterogeneous ribonucleoparticle (hnRNP)-K correspond to the major cellular poly(rC)-binding proteins. mRNA for both PCBPs were detected in all the human tissues analysed. Both proteins contain three K-homologous (KH) domains which share similarity with other KH domain proteins, including the fragile-X protein FMR1, and which are positioned as in hnRNP-K and nova, i.e. with two closely spaced domains at the N-terminus and one at the C-terminus. PCBPs do not contain RGG boxes or any other known nucleic-acid-binding motifs. Expression in the vaccinia virus system showed that both proteins are post-translationally modified in vivo, a fact that was confirmed by [32P]orthophosphate labelling. Northwestern-blot analysis showed that the non-phosphorylated forms bind tenaciously to poly(rC) in vitro, while significantly less binding was observed for the phosphorylated variants. Escherichia coli expressed proteins also bound poly(rG), albeit at a lower level. In addition, PCBP-2 bound poly(rU), whereas very little binding to poly(rA) was observed for both proteins.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 none-quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications, 890 polypeptides have been identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v) vaccinia virus expression of full length cDNAs. These are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore, we list 239 microsequenced proteins recorded in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and, ultimately, to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Acidic nuclear proteins (M(r) between 64,000 and 66,000; pI 4.9 to 5.5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1 and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle. Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.

The master two-dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post-translational modifications. 763 proteins have been identified (protein name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP-binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA mapping and sequence information and that offer an integrated approach to the study of gene expression. With the integrated approach offered by two-dimensional gel protein databases it is now possible to reveal phenotype-specific protein(s), to microsequence them, to search for homology with previous identified proteins, to clone the cDNAs, to assign partial protein sequences to genes for which the full DNA sequence and the chromosome location are known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Comprehensive two-dimensional gel protein databases will provide an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways, and cytoskeletal systems, both under physiological and abnormal conditions, and are expected to lead to the identification of new regulatory networks. So far, about 20% (600 out of 2,980) of the total number of proteins recorded in the human keratinocyte protein database have been identified and we are actively gathering qualitative and quantitative biological data on all resolved proteins. Given the current improvements on microsequencing as well as the availability of specific antibodies, it seems feasible to expect that most known keratinocyte proteins will be identified in the very near future. This feast will reveal a wealth of new proteins that will become amenable to experimentation both at the biochemical and molecular biology level.

The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease.

The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus under normal running conditions and (ii) low-abundant proteins that can only be detected after prolonged gel exposure. Annotation categories updated in this version include "protein name", "antibody against protein", "cellular localization", and "microsequenced proteins". New entries include "human autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various cellular functions both under physiological and abnormal conditions.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.

Analysis of the protein patterns of normal and psoriatic noncultured unfractionated keratinocytes has revealed several low-molecular-weight proteins that are highly up-regulated in psoriatic epidermis. Here, we have cloned and sequenced the cDNA (clone 1085) for one of these proteins that we have termed psoriasin. The deduced sequence predicted a protein of molecular weight of 11,457 daltons and a pI of 6.77. The protein co-migrated with psoriasin as determined by two-dimensional (2D) gel analysis of [35S]-methionine-labeled proteins expressed by RK13 cells transfected with clone 1085 using the vaccinia virus expression system. Analysis of the predicted sequence revealed a potential calcium-binding sequence of the EF-hand type, as well as the absence of a signal sequence at its amino terminal. Psoriasin is not related to other proteins that migrate closely in 2D gels (MRP 14, also known as calgranulin B, L1 and calprotectin; MRP 8, or calgranulin A and cystatin A or stefin A), and bears no significant sequence homology with any other protein of known primary structure. Increased expression of psoriasin mRNA in psoriatic keratinocytes was confirmed by Northern blotting and in situ hybridization. Psoriasin showed a restricted occurrence in fetal human tissues as determined by 2D gel electrophoresis. Of 21 tissues analyzed, only ear, skin, and tongue showed significant levels of this protein. Psoriasin was not detected in normal human fibroblasts, lymphocytes, endothelial cells and transformed epithelial cells of keratinocyte origin. Granulocyte extracts contained this protein suggesting that its overexpression by psoriatic keratinocytes may be linked to the inflammatory stimuli.

Analysis of cellular protein patterns by computer-aided 2-dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2-dimensional gel protein databases that may link protein and DNA information and that offer a global approach to the study of the cell. Using the integrated approach offered by 2-dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2-dimensional gel protein databases are becoming increasingly important in view of the concerted effort to map and sequence the entire genome.

A new version of the MRC-5 two-dimensional gel cellular protein database (Celis et al., Electrophoresis 1989, 10, 76-115) is presented. Gels were scanned with a Molecular Dynamics laser scanner and processed by the PDQUEST II software. A total of 1895 [35S]methionine-labeled cellular polypeptides (1323 with isoelectric focusing and 572 with nonequilibrium pH gradient electrophoresis) are recorded in this database, containing quantitative and qualitative data on the relative abundance of cellular proteins synthesized by quiescent, proliferating and SV40 transformed MRC-5 fibroblasts. Of the 592 proteins quantitated so far, the levels of 138 were up- or down-regulated (51 and 87, respectively) by two times or more in the transformed cells as compared to their normal proliferating counterparts, while only 14 behaved similarly in quiescent cells. Seven MRC-5 SV40 proteins, including plastin and two interferon-induced proteins, were not detected in the master MRC-5 images. The identity of 36 of the transformation-sensitive proteins whose levels are up or down regulated by two times or more was determined and additional information can be transferred from the master transformed human epithelial amnion cells (AMA) database (Celis et al., Electrophoresis 1990, 11, 989-1071) for those polypeptides of known and unknown identity that have been matched to AMA polypeptides. As more information is gathered in this and other laboratories, including data on oncogene proteins and transcription factors, this comprehensive database will outline an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways and cytoskeletal systems that may be directly or indirectly involved in properties associated with the transformed state.

A total of 3430 polypeptides (2592 cellular; 838 secreted) from transformed human amnion cells (AMA) labeled with [35S]methionine were separated and recorded using computer-aided two-dimensional (2-D) gel electrophoresis. A master 2-D gel database of cellular protein information that includes both qualitative and quantitative annotations has been established. The protein numbers in this database differ from those reported in an earlier version (Celis et al. Leukemia 1988, 2,561-602) as a result of changes in the scanning hardware. The reported information includes: percentage of total radioactivity recovered from the gels (based on quantitations of polypeptides labeled with a mixture of 16 14C-amino acids), protein name (including credit to investigators that aided identification), antibody against protein, cellular localization, (nuclear, 40S hnRNP, 20S snRNP U5, proteasomes, endoplasmic reticulum, mitochondria, Golgi, ribosomes, intermediate filaments, microfilaments and microtubules), levels in fetal human tissues, partial protein sequences (containing information on 48 human proteins microsequenced so far), cell cycle-regulated proteins, proteins sensitive to interferons alpha, beta, and gamma, heat shock proteins, annexins and phosphorylated proteins. The results presented should be considered as the initial phase of a joint effort between our laboratories to undertake a general and systematic analysis of human proteins. Using this integrated approach it will be possible to identify phenotype-specific proteins, to microsequence them and store the information in the database, to identify the corresponding genes, to search for homology with previously characterized proteins and to study the function of groups of proteins (pathways, organelles, etc.) that exhibit interesting regulatory properties. In particular, the 2-D gel protein database may become increasingly important in view of the concerted effort to map and sequence the entire human genome.

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.