Molecular Chaperones

The Hsp70 Super-family

Pathways For Protein Folding

Protein Folding in Cells Occurs in a Crowded Environment where Misfolding & Aggregation

are Favored

Hsp 70 Family Proteins Assist Protein Folding in an Energy Dependent Process

A. Hsp70 domain structure & Co-chaperone sites

B. Hsp70 chaperoning Cycle

The Hsp 70 Super-Family Contains Diverse Members With Related Domain Structures

Protein Folding in Eukaryotes Involves Multi-Chaperone Systems

Hsp90 assisted

Hsp60 assisted

The Hsp70 Super Family Contains Distinct Sub-Families

The C. elegans Hsp70 Chaperones

• Cytoplasmic & Nuclear– Hsp70– Hsp-1– Hsp110 homologue

• Endoplasmic Reticulum– Hsp-3– Hsp-4– Grp170 homologue

• Mitochondria– Hsp-6

• Hsp-5 is a pseudogene

Hsp 70 Family in Drosophila

Heat-shock-protein-70Ba

Heat-shock-protein-70Bb

Hsp70Bbb

Heat-shock-protein-70Bc

87B12-87B12

87B14-87B14

87B14-87B14

87B14-87B1587A3-87A3

70 kDa Hsps Locus

Hsc70-3 Heat shock protein cognate (Grp78) 87A3

Large MW Hsp70s Hsc70 Cb (hsp110) 70C-b

Hsp70 Family Members are Found in All Major Cellular Compartments

• Cytoplasm & Nucleus– Hsp70 (multiple paralogs)– Hsp110 (Multiple Paralogs)

• Mitochondria & Chloroplasts– Hsp70

• Endoplasmic Reticulum– Grp78 (hsp 70 multiple paralogs) – Grp170

Cells Respond to Stress by Increased Synthesis of Molecular Chaperones

• Misfolded proteins in the cytoplasm & nucleus induce the heat shock response, synthesis of additional heat shock proteins - Hsp’s

• Misfolded proteins in the endoplasmic reticulum induce the unfolded protein response, synthesis of additional ER chaperones - Grp’s

IRE-1

XBP-1

The Unfolded Protein Response (UPR)

• The UPR occurs when proteins are misfolded in the endoplasmic reticulum (ER).

• Reducing agents, such as DTT, interfere with disulfide bond formation while drugs can interfere with glycosylation; both agents cause proteins to misfold in the ER thus triggering the UPR.• The product of the ire-1 gene is the sensor of misfolded proteins and when activated removes an intron from the pre mRNA from the xbp-1 gene.• Active xbp-1 protein (from spliced mRNA) activates the genes that code for ER chaperones, such as hsp-4.

Hsp4 (grp78)grp170

Cellular Stresses Promote Misfolding and Aggregation of Proteins

• High temperatures

• Heavy metals

• Alcohol

• Anoxia

• Osmotic stress

• Energy starvation

• Reducing agents

The Structure of Yeast Hsp110

Hsp110 is a Nucleotide Exchange Factor for Hsp70

SummaryEfficient folding of many newly synthesized proteins depends on assistance frommolecular chaperones, which serve to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chains binding chaperones, including trigger factor, Hsp70, and prefoldin, stabilize elongating chains on ribosomes in a Non-aggregated state. Folding in the cytosol is achieved either on controlled chain release from these factors or after transfer of newly synthesized proteins to downstream chaperones, such as the chaperonins. These are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. Understanding how the thousands of different proteins synthesized in a cell use this chaperone machinery has profound implications for biotechnology and medicine. -