Substrate Proteins In Cells Research Articles

The expression and clinical significance of STAMBP in breast cancer.

Breast cancer is the leading cause of death from cancer in women worldwide. STAMBP functions as a JAMM family deubiquitinating enzyme that modulates the stability of substrate proteins in cells by cleaving ubiquitin moieties. The expression of STAMBP and its clinical significance in breast cancer remain unclear. The level of the STAMBP protein in noncancerous and tumor tissues of breast cancer patients was examined by immunohistochemical staining. The expression of STAMBP mRNA in tissues based on healthy individual and breast cancer patient data in the TCGA database was evaluated. The association between the expression of STAMBP mRNA and clinical features and prognosis was evaluated using TCGA database. Cell growth was assessed by Cell Counting Kit-8 (CCK-8) assay, and cell migration and invasion were assessed by wound healing and Transwell assays. Activation of the ERK signaling was detected by Western blotting. The expression of STAMBP was markedly upregulated in the cytoplasm of tumor cells from breast cancer patients. The level of STAMBP was closely associated with the tumor subtype and size and the TNM stage of the breast cancer patients. Importantly, high expression of STAMBP predicted poor overall survival (OS) for breast cancer patients. Furthermore, knockdown of STAMBP expression reduced cell mobility and invasion of breast cancer cells. Notably, the phosphorylation of EGFR and ERK was markedly reduced in STAMBP-knockdown cells. STAMBP plays a critical role in the progression of breast cancer and may serve as a biomarker to monitor the progression of the disease.

Heat shock-induced chaperoning by Hsp70 is enabled in-cell.

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Proteomic Identification of Protein Tyrosine Phosphatase and Substrate Interactions in Living Mammalian Cells by Genetic Encoding of Irreversible Enzyme Inhibitors.

Protein tyrosine phosphatases (PTPs) play critical roles in cell signaling pathways, but identification of unknown PTPs for a given substrate in live cells remain technically challenging. Here, we synthesized a series of tyrosine-based irreversible PTP inhibitors and characterized by site-specific encoding on substrate proteins in cells with an expanded genetic code. By fine-tuning the chemical reactivity, we identified optimal active amino acid probes to covalently cross-link a PTP and its substrate both in vitro and in mammalian cells. Using HER2 as an example, we provide first direct evidence of HER2 Y1023 and SHP2 cross-linking in situ in living human cells. Moreover, proteomic analysis using our approach identified PTP1B as a novel phosphatase for HER2 that specifically dephosphorylated pY1221 position, which may shed light on the puzzle of PTP1B's role in HER2 positive breast cancer. This novel method provides a useful tool for dissecting tyrosine phosphoregulation in living cells.

DegP functions as a critical protease for bacterial acid resistance.

To counteract the lethal acid stress, bacteria explore such strategies as cytoplasmic decarboxylase-catalyzed proton consumption and periplasmic chaperone-assisted protein refolding. Here, we report a periplasmic protease-mediated acid resistance mechanism in Escherichiacoli. Deletion of the protease gene degP dramatically decreases the viability of late log or early stationary phase cells against the extreme acid stress (pH 2.3), which can only be minimally rescued by complementary expression of the protease-deficient DegP(S210A) mutant protein. Similarly, DegQ, a homolog of DegP, also contributes to the bacterial acid resistance, but SurA as an important periplasmic chaperone hardly exhibits protection effect. Invitro studies reveal that DegP completely loses its protease activity under acidic condition but is able to partially reactivate upon neutralization. Importantly, we demonstrate the interaction of DegP with typical cellular substrate proteins in cells during acid stress and/or recovery stages by using unnatural amino acid-mediated invivo photo-crosslinking, as well as the degradation of periplasmic proteins by DegP during recovery after acidic denaturation. These data illustrate the role of DegP in bacterial acid resistance conceivably via degrading those acid-induced misfolded proteins. Our findings, together with earlier reports, suggest a comprehensive acid resistance strategy adopted by bacteria such that in the ATP-deficient extra-cytoplasm, the inevitable misfolded proteins induced by acid stress are refolded by a chaperone (e.g., HdeA/HdeB) and/or cleaved by a protease (e.g., DegP/DegQ) while in the cytoplasm excessive protons are directly consumed or exported.

Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation.

Investigations of the molecular processes that sustain life must include studies of these processes in their normal cellular environment. The bimolecular fluorescence complementation (BiFC) assay provides an approach for the visualization of protein interactions and modifications in living cells. This assay is based on the facilitated association of complementary fragments of a fluorescent protein that are fused to interaction partners. Complex formation by the interaction partners tethers the fluorescent protein fragments in proximity to each other, which can facilitate their association. The BiFC assay enables sensitive visualization of protein complexes with high spatial resolution. The temporal resolution of BiFC analysis is limited by the time required for fluorophore formation, as well as the stabilization of complexes by association of the fluorescent protein fragments. Many modifications and enhancements to the BiFC assay have been developed. The multicolor BiFC assay enables simultaneous visualization of multiple protein complexes in the same cell, and can be used to investigate competition among mutually exclusive interaction partners for complex formation in cells. The ubiquitin-mediated fluorescence complementation (UbFC) assay enables visualization of covalent ubiquitin family peptide conjugation to substrate proteins in cells. The BiFC assay can also be used to visualize protein binding to specific chromatin domains, as well as other molecular scaffolds in cells. BiFC analysis therefore provides a powerful approach for the visualization of a variety of processes that affect molecular proximity in living cells. The visualization of macromolecular interactions and modifications is of great importance owing to the central roles of proteins, nucleic acids and other macromolecular complexes in the regulation of cellular functions. This tutorial review describes the BiFC assay, and discusses the advantages and disadvantages of this experimental approach. The review will be of interest to scientists interested in the investigation of macromolecular interactions or modifications who need exquisite sensitivity for the detection of their complexes or conjugates of interest.

A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.

Systematic Identification and Analysis of Mammalian Small Ubiquitin-like Modifier Substrates

Small ubiquitin-like modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an in vitro expression cloning approach, we have identified 40 human SUMO1 substrates. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. We have validated the sumoylation of 24 substrates in living cells. Analysis of this panel of SUMO substrates leads to the following observations. 1) Sumoylation is more efficient in vitro than in living cells. Polysumoylation occurs on several substrates in vitro. 2) SUMO isopeptidases have little substrate specificity. 3) The SUMO ligases, PIAS1 and PIASxbeta, have broader substrate specificities than does PIASy. 4) Although SUMO1 and SUMO2 are equally efficiently conjugated to a given substrate in vitro, SUMO1 conjugation is more efficient in vivo. 5) Most SUMO substrates localize to the nucleus, and sumoylation does not generally affect their subcellular localization. Therefore, sumoylation appears to regulate the functions of its substrates through multiple, context-dependent mechanisms.