scholarly journals Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

1993 ◽  
Vol 123 (1) ◽  
pp. 119-126 ◽  
Author(s):  
W Voos ◽  
B D Gambill ◽  
B Guiard ◽  
N Pfanner ◽  
E A Craig
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Dual Role ◽  
Intermembrane Space ◽  
Heme Binding ◽  
Membrane Translocation ◽  
Amino Terminal ◽  
The Matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

scholarly journals A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.

1993 ◽  
Vol 123 (1) ◽  
pp. 109-117 ◽  
Author(s):  
B D Gambill ◽  
W Voos ◽  
P J Kang ◽  
B Miao ◽  
T Langer ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Polypeptide Chain ◽  
Dual Role ◽  
Temperature Sensitive ◽  
Membrane Translocation ◽  
Mitochondrial Membranes ◽  
Inner Membrane ◽  
Amino Terminal ◽  
The Matrix

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

scholarly journals The Amino-terminal Domain of Heat Shock Protein 90 (hsp90) That Binds Geldanamycin Is an ATP/ADP Switch Domain That Regulates hsp90 Conformation

1997 ◽  
Vol 272 (38) ◽  
pp. 23843-23850 ◽  
Author(s):  
James P. Grenert ◽  
William P. Sullivan ◽  
Patrick Fadden ◽  
Timothy A. J. Haystead ◽  
Jenny Clark ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals In Vivo Cytotoxic T Lymphocyte Elicitation by Mycobacterial Heat Shock Protein 70 Fusion Proteins Maps to a Discrete Domain and Is Cd4+ T Cell Independent

2000 ◽  
Vol 191 (2) ◽  
pp. 403-408 ◽  
Author(s):  
Qian Huang ◽  
Joan F.L. Richmond ◽  
Kimiko Suzue ◽  
Herman N. Eisen ◽  
Richard A. Young
Keyword(s):  
T Cell ◽  
Heat Shock ◽  
T Lymphocytes ◽  
Heat Shock Protein ◽  
Fusion Proteins ◽  
Cytotoxic T Lymphocyte ◽  
Protein Domain ◽  
Cd4 T Cell ◽  
Fusion Partner

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8+ cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4+ T lymphocytes. We found that the ability to elicit CD8+ CTLs depends on a discrete 200–amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8+ T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8+ CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4+ T cell–deficient individuals.

scholarly journals Cyclophilin-40: evidence for a dimeric complex with hsp90

1995 ◽  
Vol 307 (1) ◽  
pp. 5-8 ◽  
Author(s):  
K Hoffmann ◽  
R E Handschumacher
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Fusion Protein ◽  
Steroid Receptors ◽  
Heat Shock Protein 90 ◽  
Binding Activity ◽  
Dimeric Complex ◽  
Glutathione S Transferase ◽  
High Concentration ◽  
Cyclophilin 40

The expression of human cyclophilin 40 (CyP-40) as a glutathione S-transferase fusion protein has provided a means to identify cellular components that are in association with this ubiquitous protein. When the fusion protein was coupled to a GSH affinity matrix, heat-shock protein 90 (hsp90) was found to be the predominant associated protein in all tissue extracts examined. The relatively high concentration of each of these proteins in various tissues indicates that the dimeric complex exists in concentrations that exceed those of the inactive steroid receptors of which each protein is a component. Association does not occur with heat-shock protein 70 and is not affected by cyclosporin A (CsA). Independent expression of two domains of CyP-40 permitted dissociation of N-terminal isomerase and CsA binding activity from the hsp90 binding site, which is located at the FKBP-59-like C-terminal region. The biological association of CyP-40 with hsp90 in many tissues may reflect a conjoint role in protein folding and trafficking.

Phase Ib study of heat shock protein 90 inhibitor, onalespib in combination with paclitaxel in patients with advanced, triple-negative breast cancer (NCT02474173).

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. TPS1127-TPS1127
Author(s):  
Robert Wesolowski ◽  
Maryam B. Lustberg ◽  
Raquel E. Reinbolt ◽  
Jeffrey Bryan VanDeusen ◽  
Sagar D. Sardesai ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Standard Dose ◽  
Heat Shock Protein 90 ◽  
Mapk Signaling ◽  
Amino Terminal ◽  
Client Proteins

TPS1127 Background: Heat shock protein 90 (HSP90) is a molecular chaperone which is necessary for proper folding and stabilization of proteins. Client proteins of HSP90 include many oncogenic proteins known to be over-activated in triple negative breast cancer such as AKT, EGFR, members of RAS/MAPK signaling pathway and androgen receptor. High expression of HSP90 in breast cancer has been associated with poor outcome. In addition, over-expression of HSP90 client proteins such as AKT and c-RAF has been implicated in paclitaxel resistance. Onalespib (AT13387) is a synthetic non-ansamycin small molecule that acts as an inhibitor of HSP90 by binding to the amino terminal of the protein and has dissociation constant (Kd) of 0.71 nM. Methods: Patients with inoperable or metastatic, triple negative or < 10% hormone receptor positive breast cancer are treated with onalespib and paclitaxel on days 1, 8, 15 every 28 days. Paclitaxel is given at a standard dose of 80 mg/m2 while the dose of onalespib is gradually increased using standard 3+3 design (see table). In order to assess the effect of each drug on pharmacokinetics of the other drug, onalespib is given on day -7 prior to cycle 1 and skipped on day 1 of cycle 1 during which paclitaxel is administered alone. The primary objective of the study is to determine recommended phase II dose and assess the toxicity profile of the combination. The secondary objectives include pharmacokinetic of each agent. Overall response rate, response duration and progression-free survival will also be assessed. The study is currently enrolling patients to dose level 2. Clinical trial information: NCT02474173. [Table: see text]

Sub-lethal heat shock induces plasma membrane translocation of 70-kDa heat shock protein in viable, but not in apoptotic, U-937 leukaemia cells

Apmis ◽  
2010 ◽  
Vol 118 (3) ◽  
pp. 179-187 ◽  
Author(s):  
ELENA B. LASUNSKAIA ◽  
IRINA FRIDLIANSKAIA ◽  
ANDREA V. ARNHOLDT ◽  
MILTON KANASHIRO ◽  
IRINA GUZHOVA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Membrane Translocation

scholarly journals Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

1999 ◽  
Vol 10 (7) ◽  
pp. 2461-2474 ◽  
Author(s):  
Martin Kurz ◽  
Heiko Martin ◽  
Joachim Rassow ◽  
Nikolaus Pfanner ◽  
Michael T. Ryan
Keyword(s):  
Crossing Over ◽  
Intermembrane Space ◽  
Membrane Translocation ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Surface Receptors ◽  
Amino Terminal ◽  
Membrane Carriers ◽  
Positively Charged ◽  
Tim Proteins

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

scholarly journals Heat Shock Protein 70 of the Agent of Human Granulocytic Ehrlichiosis Binds to Borrelia burgdorferi Antibodies

1998 ◽  
Vol 5 (1) ◽  
pp. 118-120 ◽  
Author(s):  
Jacob W. Ijdo ◽  
Yan Zhang ◽  
Matthew L. Anderson ◽  
David Goldberg ◽  
Erol Fikrig
Keyword(s):  
Heat Shock ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Heat Shock Protein ◽  
Immunoblot Analysis ◽  
Test Results ◽  
Hsp 70 ◽  
Granulocytic Ehrlichiosis ◽  
Amino Terminal ◽  
Human Granulocytic Ehrlichiosis

ABSTRACT We describe a patient with human granulocytic ehrlichiosis (HGE), a diagnosis confirmed by PCR and immunoblot analysis. Unexpectedly, immunoglobulin G (IgG) directed towards an 80-kDa ehrlichial antigen (without detectable IgM) was present in the patient’s serum in the first week of illness. Lyme disease immunoblots were reactive for IgG (but not IgM), a result indicative of prior exposure to the Lyme disease spirochete. Amino-terminal sequencing revealed that the 80-kDa ehrlichial antigen was an HSP-70 homolog similar to Borrelia burgdorferi HSP-70. We conclude that antibodies against B. burgdorferi HSP-70 may cross-react with the ehrlichial heat shock protein and that this possibility must be considered when serologic test results for HGE and Lyme disease are interpreted.

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