Fas/Fas-L and its Fas/Fas-L and its involvement ininvolvement in

Autoimmune DiabetesAutoimmune DiabetesBIOL537: ImmunobiologyBIOL537: Immunobiology

Spring 2001Spring 2001Brooke MartinBrooke MartinPaul NalliahPaul Nalliah

ApoptosisApoptosis Cysteine proteases a part of the caspase Cysteine proteases a part of the caspase

protein family are activated specifically in protein family are activated specifically in apoptosisapoptosis

Caspases – proteins that selectively cleave a Caspases – proteins that selectively cleave a restricted set of target proteins at one or few restricted set of target proteins at one or few positions in the primary sequence; results in positions in the primary sequence; results in inactivation or activation of the target proteininactivation or activation of the target protein

Caspases are synthesized as enzymatically Caspases are synthesized as enzymatically inert zymogens that require proteolytic inert zymogens that require proteolytic cleavage to be activatedcleavage to be activated

Three general mechanisms Three general mechanisms of caspase activationof caspase activation

• Association with a regulatory subunit – association Association with a regulatory subunit – association with a dedicated protein cofactor, Apaf-1 (caspase-9)with a dedicated protein cofactor, Apaf-1 (caspase-9)

• Induced Proximity – upon ligand binding, death Induced Proximity – upon ligand binding, death receptors, CD-95 aggregate and form membrane-receptors, CD-95 aggregate and form membrane-bound signalling complexes which recruit several bound signalling complexes which recruit several caspase molecules, under these crowded conditions, caspase molecules, under these crowded conditions, the intrinsic protease activity of the proteins the intrinsic protease activity of the proteins mutually cleave and activate one another (caspase-mutually cleave and activate one another (caspase-8, they key initiator in the death receptor pathway)8, they key initiator in the death receptor pathway)

• Processing by an upstream caspase – activation of Processing by an upstream caspase – activation of the caspase by an already activated caspase the caspase by an already activated caspase molecule (caspase –3, -6, -7)molecule (caspase –3, -6, -7)

Death Receptor Pathway: Death Receptor Pathway: Type IType I

• Triggered by members of the death-receptor Triggered by members of the death-receptor superfamily (ex CD-95)superfamily (ex CD-95)

• Binding of CD-95 ligand to CD-95 induces a Binding of CD-95 ligand to CD-95 induces a clustering of receptors and initiates the clustering of receptors and initiates the formation of death inducing signaling complex formation of death inducing signaling complex (DISC)(DISC)

• Complex recruits multiple procaspase-8 Complex recruits multiple procaspase-8 molecules, via the Fas- associated death molecules, via the Fas- associated death domain protein (FADD), resulting in caspase-8 domain protein (FADD), resulting in caspase-8 activation through induced proximityactivation through induced proximity

Death Receptor Death Receptor Pathway:Type IIPathway:Type II• Very little DISC is formed, so the caspase Very little DISC is formed, so the caspase

cascade cannot be propagated directly but cascade cannot be propagated directly but must be amplified via the mitochondriamust be amplified via the mitochondria

• Caspase-8 cleaves Bid, which activates the Caspase-8 cleaves Bid, which activates the mitochondria mitochondria

• Activated mitochondria releases pro-apoptotic Activated mitochondria releases pro-apoptotic molecules such as cytochrome c and molecules such as cytochrome c and Smac/DIABLO which work with Apaf-1 to Smac/DIABLO which work with Apaf-1 to activate procaspase-9, which then activates activate procaspase-9, which then activates downstream caspases which induce apoptosisdownstream caspases which induce apoptosis

CTL – Cytotoxic T CTL – Cytotoxic T LymphocytesLymphocytes

Granule Exocytosis PathwayGranule Exocytosis Pathway• Contain perforin and granzyme Contain perforin and granzyme

molecules in granules that are molecules in granules that are exocytosed to extracellular space exocytosed to extracellular space when in close proximity to target cellwhen in close proximity to target cell

• Perforin and granzyme enter target Perforin and granzyme enter target cell via perforin pores inducing cell via perforin pores inducing caspase cascades that cause caspase cascades that cause apoptosisapoptosis

CTL – The FAS (CD-95) CTL – The FAS (CD-95) pathwaypathway

• Fas ligand exists as a Fas ligand exists as a transmembrane protein which transmembrane protein which connects with Fas on the target cellconnects with Fas on the target cell

• The ligation of the two trimeric Fas The ligation of the two trimeric Fas units results in the activation of the units results in the activation of the death receptor pathway resulting in death receptor pathway resulting in apoptosisapoptosis

Proposed model of target-cell apoptosis stimulated Proposed model of target-cell apoptosis stimulated by CTLsby CTLs

Applications of CD-95 in the Applications of CD-95 in the Immune SystemImmune System

• CD-95L is expressed continuously in immune-CD-95L is expressed continuously in immune-privileged sites such as the testis and the eye, which privileged sites such as the testis and the eye, which may be used as tumor suppressors or altered to may be used as tumor suppressors or altered to delay the rejection of transplantsdelay the rejection of transplants

• Viral gene products of HIV-infected cells penetrate Viral gene products of HIV-infected cells penetrate non-infected cells in the immune system, especially non-infected cells in the immune system, especially CD+4 cells, rendering them hypersensitive to CD+4 cells, rendering them hypersensitive to apoptosis via CD-95. This suggests that CD-95 may apoptosis via CD-95. This suggests that CD-95 may have a significant role in the T helper cell depletion of have a significant role in the T helper cell depletion of AIDS. This information could prove useful in the AIDS. This information could prove useful in the development of therapeutic strategies aimed at development of therapeutic strategies aimed at intervening with the loss of CD+4 cells in HIV positive intervening with the loss of CD+4 cells in HIV positive individuals.individuals.