Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system

Hepadnaviruses are DNA viruses that replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase (RT), P protein. DNA synthesis from the pregenomic RNA is initiated by binding of P to the ε signal. Using ε as template and a Tyr-residue for initiation, the RT synthesizes a DNA oligo (priming) as primer for full-length DNA. Priming strictly requires prior RT activation by chaperones. Active P–ε complexes have been reconstituted in vitro, but whether in addition to the heat-shock protein 70 (Hsp70) system the Hsp90 system is essential has been controversial. Here we quantitatively compared Hsp70 versus Hsp70 plus Hsp90 RT activation, and corroborated that the Hsp70 system alone is sufficient; however, Hsp90 as well the Hsp70 nucleotide exchange factor Bag-1 markedly stimulated activation by increasing the steady-state concentration of the activated metastable RT form P*, though by different mechanisms. Hsp90 inhibition in intact cells by geldanamycin analogs blocked hepadnavirus replication, however not completely and only at severely cytotoxic inhibitor concentrations. While compatible with a basal level of Hsp90 independent in vivo replication, unambiguous statements are precluded by the simultaneous massive upregulation of Hsp70 and Hsp90

Regulation of Hsp90 and its substrate p53

Das molekulare Chaperone Hsp90 ist eine ATP-abhängigen Maschine, die in der Zelle über Co-Faktoren und post-translationalen Modifikationen reguliert wird. Wie genau diese Regulation aussieht wurde für Aha1, den bisher einzig bekannten Aktivator von Hsp90 detailliert aufgeklärt. Eine weitere Regulation von Hsp90 erfolgt in vivo durch die S-Nitrosylierung eines konservierten Cysteins. Welche Auswirkungen sich aus der Veränderung dieser spezifischen Aminosäureseitenkette auf die Aktivität von Hsp90 ergeben, wurde über eine Struktur-Funktionsanalyse aufgeklärt. Über die Regulation von Hsp90 kann wiederum die Aktivität seiner Substrate beeinflusst werden. Hierzu wurde die Wechselwirkung von Hsp90 und dem Tumorsuppressor p53 untersucht und ein Bindungsmodell erstellt. Die Struktur-Funktionsbeziehung von p53 durch inter-molekulare Wechselwirkungen wurde ebenfalls untersucht.The molecular chaperone Hsp90 is an ATP-dependent machinery, which is regulated by different co-factors and post-translational modifications. This regulation is characterized for Aha1, an unique activator of Hsp90. Another type of the Hsp90 regulation in vivo is based on the S-nitrosylation of a conserved cysteine residue. The impact on the activity of Hsp90 by the modification of this specific amino acid side chain is characterized for the structure-function relationship of Hsp90. The regulation of Hsp90 affects the activity of its substrates. Here, a detailed binding model for the interaction of Hsp90 with the tumor suppressor protein p53 is proposed, and additionally, the structure-function relationship of p53 defined by inter-molecular interactions is characterized

Grundschutz im Klinikum - Praxisbericht zum Einsatz von IT-Grundschutz an einem bayerischen Universitätsklinikum.

Eignet sich die IT-Grundschutz-Systematik des BSI auch im medizininformatischen Umfeld? Ein Praxistest an einem bayerischen Universitätsklinikum hat das ergründet und die Methodik mit dem GSTOOL bis zum Basis-Sicherheitscheck 1 umgesetzt

Hsp90 is regulated by a switch point in the C-terminal domain

Heat shock protein 90 (Hsp90) is an abundant, dimeric ATP-dependent molecular chaperone, and ATPase activity is essential for its in vivo functions. S-nitrosylation of a residue located in the carboxy-terminal domain has been shown to affect Hsp90 activity in vivo. To understand how variation of a specific amino acid far away from the amino-terminal ATP-binding site regulates Hsp90 functions, we mutated the corresponding residue and analysed yeast and human Hsp90 variants both in vivo and in vitro. Here, we show that this residue is a conserved, strong regulator of Hsp90 functions, including ATP hydrolysis and chaperone activity. Unexpectedly, the variants alter both the C-terminal and N-terminal association properties of Hsp90, and shift its conformational equilibrium within the ATPase cycle. Thus, S-nitrosylation of this residue allows the fast and efficient fine regulation of Hsp90