detection-detergent-sensitive-interactions-between-membrane

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Boston University School of Medicine

Novel classes of anticancer drugs, including proteasome inhibitors and Hsp90 inhibitors, potently induce heat shock proteins (Hsps). Because Hsps show antiapoptotic activities, we suggested that suppression of such induction may sensitize cancer cells to these drugs. Here, we knocked out the major heat shock transcription factor HSF-1 in several cancer cell lines using small interfering RNA and showed that such cells, which can no longer induce Hsps in response to proteasome and Hsp90 inhibitors, become more sensitive to these drugs. Furthermore, we developed a high-throughput screen for small molecules that inhibit induction of Hsps. The first step was a cell-based screen for inhibitors of Hsps-mediated luciferase refolding followed by a counterscreen for toxicity. The second step was a direct testing for inhibition of Hsp induction by immunoblotting with anti-Hsp72 antibody. After screening of 20,000 compounds from several diversity libraries, we focused on a compound we called NZ28, which potently inhibited induction of Hsps by heat shock, proteasome, and Hsp90 inhibitors in a variety of cell lines, and showed no significant toxicity. After testing of a set of analogues of NZ28, we identified a structural element that was critical for the activity. We also identified another inhibitor of the Hsp induction that was practically nontoxic. This compound, which we called emunin, strongly sensitized myeloma cells to proteasome and Hsp90 inhibitors and prostate carcinoma cells to proteasome inhibitors. This work indicates that targeting the heat shock response may facilitate use of proteasome and Hsp90 inhibitors for cancer treatment.

Targeting heat shock response to sensitize cancer cells to proteasome and Hsp90 inhibitors.

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.

Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery.

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein-RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.

Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

Sortilin is a multiligand sorting receptor responsible for the anterograde transport of lysosomal enzymes and substrates. Here we demonstrate that sortilin is also involved in retrograde protein traffic. In cultured 3T3-L1 adipocytes, sortilin together with retromer rescues Glut4 from degradation in lysosomes and retrieves it to the TGN, where insulin--responsive vesicles are formed. Mechanistically, the luminal Vps10p domain of sortilin interacts with the first luminal loop of Glut4, and the cytoplasmic tail of sortilin binds to retromer. Ablation of the retromer does not affect insulin signaling but decreases the stability of sortilin and Glut4 and blocks their entry into the small vesicular carriers. As a result, Glut4 cannot reach the insulin-responsive compartment, and insulin-stimulated glucose uptake in adipocytes is suppressed. We suggest that sortilin- and retromer-mediated Glut4 retrieval from endosomes may represent a step in the Glut4 pathway vulnerable to the development of insulin resistance and diabetes.

Nava Zaarur

Department of Biochemistry

Boston University School of Medicine

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Nava Zaarur

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry

Boston University School of Medicine

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Department of Biochemistry