Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

Ice as a matrix for IR-matrix-assisted laser desorption/ionization: mass spectra from a protein single crystal.

Lasers emitting in the ultraviolet wavelength range of 260-360 nm are almost exclusively used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of macromolecules. Reports about the use of lasers emitting in the infrared first appeared in 1990/1991. In contrast to MALDI in the ultraviolet, a very limited number of reports on IR-MALDI have since been published. Several matrices have been identified for infrared MALDI yielding spectra of a quality comparable to those obtained in the ultraviolet. Water (ice) was recognized early as a potential matrix because of its strong O-H stretching mode near 3 microm. Interest in water as matrix derives primarily from the fact that it is the major constituent of most biological tissues. If functional as matrix, it might allow the in situ analysis of macromolecular constituents in frozen cell sections without extraction or exchanging the water. We present results that show that IR-MALDI of lyophilized proteins, air dried protein solutions, or protein crystals up to a molecular mass of 30 kDa is possible without the addition of any separate matrix. Samples must be frozen to retain a sufficient fraction of the water of hydration in the vacuum. The limited current sensitivity, requiring at least 10 pmol of protein for a successful analysis needs to be further improved.