We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals.

The gene cul-1 (formerly lin-19) is a negative regulator of the cell cycle in C. elegans. Null mutations cause hyperplasia of all tissues. cul-1 is required for developmentally programmed transitions from the G1 phase of the cell cycle to the GO phase or the apoptotic pathway. Moreover, the mutant phenotype suggests that G1-to-S phase progression is accelerated, overriding mechanisms for mitotic arrest and producing abnormally small cells. Significantly, diverse aspects of cell fate and differentiation are unaffected in cul-1 mutants. cul-1 represents a conserved family of genes, designated cullins, with at least five members in nematodes, six in humans, and three in budding yeast.

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Members of the ICE/ced-3 gene family have been implicated as components of the cell death pathway. Based on similarities with the structural prototype interleukin-1␤-converting enzyme (ICE), family members are synthesized as proenzymes that are proteolytically processed to form active heterodimeric enzymes. In this report, we describe a novel member of this growing gene family, ICE-LAP3, which is closely related to the death effector Yama/CPP32/Apopain. Pro-ICE-LAP3 is a 35-kDa protein localized to the cytoplasm and expressed in a variety of tissues and cell lines. Overexpression of a truncated version of ICE-LAP3 (missing the pro-domain) induces apoptosis in MCF7 breast carcinoma cells. Importantly, upon receipt of a death stimulus, endogenous ICE-LAP3 is processed to its subunit forms, suggesting a physiological role in cell death. This is the first report to demonstrate processing of a native ICE/ ced-3 family member during execution of the death program and the first description of the subcellular localization of an ICE/ced-3 family member.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/ CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGG pentapeptide, rather than the QACRG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.Apoptosis, or programmed cell death, is a physiologic process important in the normal development and homeostasis of metazoans (1). It is becoming apparent that a class of cysteine proteases homologous to Caenorhabditis elegans Ced-3 play the role of "executioner" in the apoptotic mechanism (2-4). In the nematode, two proteins, encoded by ced-3 and ced-4, are required for all somatic cell deaths that occur during development (5). Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that normally die during C. elegans embryogenesis (6). While no mammalian homologs of ced-4 have been identified, ced-3 shares sequence similarity with interleukin-1␤ converting enzyme (ICE) 1 (7), a cysteine protease involved in the processing and activation of pro-interleukin-1␤ to an active cytokine (8, 9). Recently, numerous homologs of ICE/ Ced-3 have been characterized, comprising a new gene family of cysteine proteases.To date, seven members of the ICE/Ced-3 family have been identified and include ICE (8), TX/ICH2/ICE rel-II (10 -12), ICE rel-III (10), ICH1/Nedd-2 (13, 14), Yama/CPP32/Apopain (15-17), Mch2 (16), and ICE-LAP3/Mch3/CMH-1 (18 -20). All family members share sequence homology with ICE/Ced-3 and contain an active site QACRG pentapeptide in which the cysteine residue is catalytic. Ectopic expression of these proteases in a variety of cells causes apoptosis. Phylogenetic analysis of the ICE/Ced-3 3gene family revealed three subfamilies (3, 18). Yama, ICE-LAP3, and Mch2 are closely related to C. elegans Ced-3 and comprise the Ced-3 subfamily. ICE and the ICErelated genes, ICE rel-II, and ICE rel-III form the ICE subfamily, while ICH1 and its mouse homologue, NEDD-2 form the NEDD-2 subfamily.Based on similarities with the structural prototype interleukin-1␤ converting enzyme, ICE/Ced-3 family members are synthesized as zymogens that are capable of being processed to form active heterodimeric enzymes (9). It will be important to determine which family members are in fact activated in response to apoptotic stimuli. Previous studies have demonstrated that pro-Yama and pro-ICE-LAP3 are processed into active subunits in response to various death stimuli including engagement of Fas/APO-1 or treatment w...