Alisa G. Woods

Investigation of stable and transient protein–protein interactions: Past, present, and future

This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein–protein interactions (PPIs) with a focus on their function within cells, organs, and organisms. The significance of PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis‐based and MS‐based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Heparan sulfate and chondroitin sulfate glycosaminoglycan attenuate β-amyloid(25-35) induced neurodegeneration in cultured hippocampal neurons

beta-Amyloid peptide has been reported to be toxic to neurons in vitro and in vivo. The fragment of the beta 1-42 peptide believed to be responsible for this toxicity consists of amino acids 25 to 35. beta-amyloid protein, heparan sulfate (HS) glycosaminoglycan (GAG), and proteoglycan (PG) are all localized throughout the senile plaques found in Alzheimers disease. Chondroitin sulfate (CS) and dermatan sulfate have also been found at the periphery of senile plaques. We have found that both HS and CS prevented neurite fragmentation and toxicity normally induced by beta 25-35. HS and CS by themselves did not have a significant influence on cell viability, indicating that their protective actions were not due to a general trophic effect. In contrast, cultures treated with HS and beta 1-42 did not show significantly reduced toxicity compared to cultures treated with beta 1-42 alone despite specific binding interactions. These data indicate that one function of GAGs in the brain may be to protect neurons from select toxic insults and injury, and additionally suggest that HS interacts differently with different beta-amyloid fragments. These data further suggest that different beta-amyloid fragments may induce distinct mechanisms of toxicity in vitro.

Protein–protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches

Following the sequencing of the human genome and many other organisms, research on protein-coding genes and their functions (functional genomics) has intensified. Subsequently, with the observation that proteins are indeed the molecular effectors of most cellular processes, the discipline of proteomics was born. Clearly, proteins do not function as single entities but rather as a dynamic network of team players that have to communicate. Though genetic (yeast two-hybrid Y2H) and biochemical methods (co-immunoprecipitation Co-IP, affinity purification AP) were the methods of choice at the beginning of the study of protein–protein interactions (PPI), in more recent years there has been a shift towards proteomics-based methods and bioinformatics-based approaches. In this review, we first describe in depth PPIs and we make a strong case as to why unraveling the interactome is the next challenge in the field of proteomics. Furthermore, classical methods of investigation of PPIs and structure-based bioinformatics approaches are presented. The greatest emphasis is placed on proteomic methods, especially native techniques that were recently developed and that have been shown to be reliable. Finally, we point out the limitations of these methods and the need to set up a standard for the validation of PPI experiments.

Potential biomarkers in psychiatry: focus on the cholesterol system.

● Introduction ● Methods for proteomic analysis – Sample fractionation/biochemical fractionation – MS ● The cholesterol system – Cholesterol and apolipoproteins – Cholesterol – Apos – ApoE – ApoB – ApoA1 and ApoA4 ● The cholesterol system and specific disorders – Alzheimers disease – Schizophrenia – Depression – Developmental disorders: ASDs ● Discussion – Proteomic considerations for analysis of Apos – Considering diet and lifestyle effects on the cholesterol system – Consequences of disturbed cholesterol and Apos in the CNS ● Conclusion

Astroglial ciliary neurotrophic factor mRNA expression is increased in fields of axonal sprouting in deafferented hippocampus

Evidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in the periphery raises the possibility that this factor is involved in reactive axonal growth in the brain. In situ hybridization was used in the present study to determine whether ciliary neurotrophic factor mRNA expression is increased in association with axonal sprouting in deafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNA labeling density was high in the olfactory nerve, pia mater, and aspects of the ventricular ependyma and was relatively low within areas of white matter (fimbria, internal capsule) and select neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion, hybridization was markedly increased in fields of anterograde degeneration, including most prominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratum lacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, was maximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstrated that, in both control and experimental tissue, ciliary neurotrophic factor mRNA was colocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was not colocalized with markers for oligodendrocytes or microglia. These results demonstrate that astroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminal degeneration and that increased expression is coincident with 1) increased insulin‐like growth factor‐1 and basic fibroblast growth factor expression and 2) the onset of reactive axonal growth. The synchronous expression of these glial factors in fields of deafferentation suggests the possibility of additive or synergistic interactions in the coordination of central axonal growth. J. Comp. Neurol. 386:137–148, 1997.

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

Identification of Potential Tumor Differentiation Factor (TDF) Receptor from Steroid-responsive and Steroid-resistant Breast Cancer Cells

Background: Tumor differentiation factor (TDF) is a newly identified pituitary protein with no known receptor. Results: Heat shock 70-kDa proteins are potential TDF receptor candidates. Conclusion: TDF acts on breast cells through a novel pathway. Significance: These data may help to elucidate the role of TDF. Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Identification of consistent alkylation of cysteine-less peptides in a proteomics experiment.

In a proteomics experiment, reduction and alkylation of proteins prior to enzymatic digestion ensures high sequence coverage of that protein during a database search. However, the alkylation procedure uses an excess of an alkylating agent such as iodoacetamide (IAA). Therefore, although other amino acids are alkylated, these modified peptides are not identified in a database search. Here we show that a large proportion of peptides are mono- and di-alkylated by IAA and therefore not identified via a database search. The first alkylation consistently takes place at the N-terminal amino acid. Therefore, we propose that during the database search conducted during a proteomics experiment, one should have the option of searching for any alkylated peptide at the N-terminal amino acid.

CNTF and CNTF receptor alpha are constitutively expressed by astrocytes in the mouse brain.

Ciliary neurotrophic factor (CNTF) is regarded as one of the signals that lead to astrocyte activation following central nervous system lesion. However, it is not clear if CNTF can directly initiate astrocytic responses to injury because CNTF levels are very low in most areas of the unlesioned brain and CNTF receptor (CNTFRα) expression by astrocytes has not yet been demonstrated in the intact brain. In the present study, the expression patterns of CNTF protein and CNTFRα mRNA were studied in the intact mouse brain using immunocytochemistry and in situ hybridization, respectively. These procedures were combined with immunocytochemistry for glial fibrillary acidic protein in order to identify CNTF‐ and CNTFRα‐expressing astrocytes. CNTF‐immunoreactive astrocytes were exclusively found in white matter structures such as the optical tract, the corticospinal tract, and the fimbria‐fornix. Gray matter astrocytes did not exhibit CNTF immunoreactivity. In contrast, CNTFRα mRNA‐expressing astrocytes were found in gray matter areas, preferentially in the molecular layers of the cortex and hippocampus. White matter astrocytes did not show a detectable CNTFRα mRNA signal. These data demonstrate that both CNTF and its receptor are constitutively expressed by astrocytes in mouse brain. The nonoverlapping locations of astrocytes expressing detectable levels of CNTF and CNTFRα, respectively, may be related to distinct postlesional functions of these two glial cell populations. GLIA 37:374–378, 2002.

Automated Mass Spectrometry-Based Functional Assay for the Routine Analysis of the Secretome

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.