Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.     

SIMPLE/LITAF expression induces the translocation of the ubiquitin ligase itch towards the lysosomal compartments

LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.     

LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症患者的突变分析

为了分析LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT)的突变特点, 文章分别应用PCR结合DNA序列分析方法和PCR-单链构象多态性(PCR-SSCP)结合DNA序列分析方法对6个常染色体显性遗传家系先证者和27个散发病例进行LITAF和RAB7基因突变分析; 应用PCR-SSCP结合DNA序列分析方法对14个常染色体遗传的CMT家系先证者和27个散发患者进行LMNA和MTMR2基因突变分析。结果发现: LITAF基因c.269G→A、c.274A→G序列变异和LMNA基因c.1243G→A、c.1910C→T序列变异, 未发现RAB7和MTMR2基因的序列变异。其中LITAF基因c.269G→A、LMNA基因c.1243G→A和c.1910C→T为新发现的单核苷酸多态; LITAF基因c.274A→G为已知多态。说明LITAF、RAB7、LMNA和MTMR2基因突变在中国人CMT患者中罕见。     

Mutation of SIMPLE in Charcot–Marie–Tooth 1C alters production of exosomes

Charcot–Marie–Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis.     

The topology,structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C

Background

Mutations in Lipopolysaccharide-induced tumour necrosis factor-α factor (LITAF) cause the autosomal dominant inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1C (CMT1C). LITAF encodes a 17 kDa protein containing an N-terminal proline-rich region followed by an evolutionarily-conserved C-terminal ‘LITAF domain’, which contains all reported CMT1C-associated pathogenic mutations.

Results

Here, we report the first structural characterisation of LITAF using biochemical, cell biological, biophysical and NMR spectroscopic approaches. Our structural model demonstrates that LITAF is a monotopic zinc-binding membrane protein that embeds into intracellular membranes via a predicted hydrophobic, in-plane, helical anchor located within the LITAF domain. We show that specific residues within the LITAF domain interact with phosphoethanolamine (PE) head groups, and that the introduction of the V144M CMT1C-associated pathogenic mutation leads to protein aggregation in the presence of PE.

Conclusions

In addition to the structural characterisation of LITAF, these data lead us to propose that an aberrant LITAF-PE interaction on the surface of intracellular membranes contributes to the molecular pathogenesis that underlies this currently incurable disease.

     

Cutting edge: impaired glycosphingolipid trafficking and NKT cell development in mice lacking Niemann-Pick type C1 protein

Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Valpha14-Jalpha18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent alpha-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Valpha14-Jalpha18 NKT cells.     

Distinct endosomal compartments in early trafficking of low density lipoprotein-derived cholesterol

We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013-4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5-10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.     

The heme-globin and dimerization equilibria of recombinant human hemoglobins carrying site-specific beta chains mutations

The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.     

Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor.

A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT1) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT(1) receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein-coupled receptor (GPCR) activation.     

Rab GTPases regulating receptor trafficking at the late endosome–lysosome membranes

Lysosomes serve key degradative functions for the turnover of membrane lipids and protein components. Its biogenesis is principally dependent on exocytic traffic from the late endosome via the trans‐Golgi network, and it also receives cargo to be degraded from the endocytic pathway. Membrane trafficking to the late endosome–lysosome is tightly regulated to maintain the amplitude of signalling events and cellular homeostasis. Key coordinators of lysosomal traffic include members of the Rab small GTPase family. Amongst these, Rab7, Rab9 and the more recently studied Rab22B/31 have all been reported to regulate membrane trafficking processed at the late endosome–lysosome system. We discuss what is known about the roles of these Rab proteins and their interacting partners on the regulation of traffic of important receptor proteins such as the epidermal growth factor receptor (EGFR) and the mannose 6‐phosphate receptor (M6PR), in association with the late endosome–lysosome system. Better knowledge of EGFR and M6PR traffic in this regard may aid in understanding the pathological processes, such as oncogenic transformations associated with these receptors. Copyright © 2012 John Wiley & Sons, Ltd.     

Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.     

The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.     

A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae

The late endosome and vacuole of yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome. The late endosome is the convergence point of the biosynthetic and endocytic trafficking to the vacuole. Here, we describe a novel immunodetection screen to isolate mutants defective in trafficking the soluble hydrolase carboxypeptidase Y (CPY) at the late endosome to vacuole interface (env mutants). Mutants exhibit vacuolar morphology and endocytosis defects as assayed by electron, fluorescent, and nomarski microscopy. In biochemical assays, they internally accumulate p2CPY in a dense membrane compartment lacking vacuolar properties yet display normal secretion phenotypes. The results suggest vacuolar morphology and function defects that are exclusively at the late endosome/vacuole interface. env mutants define five complementation groups. The first gene of the collection to be cloned, ENV1 is allelic to VPS35 whose established function is in retrograde trafficking from late endosome to trans-Golgi network (TGN). Microscopic, biochemical, and growth analyses establish that env1 is distinct from other alleles of VPS35 in vacuolar morphology, growth characteristics, and internal accumulation of p2CPY. Our results indicate that ENV genes may define new gene functions at the late endosome to vacuole interface.     

Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

The lipoprotein receptor-related protein-1 (LRP) adapter protein GULP mediates trafficking of the LRP ligand prosaposin, leading to sphingolipid and free cholesterol accumulation in late endosomes and impaired efflux

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands alpha2-macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.     

Rabring7, a novel Rab7 target protein with a RING finger motif

Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis.     

Effects of disulphide bridges on the activity and stability of the formate dehydrogenase from <Emphasis Type="Italic">Candida methylica</Emphasis>

Wild-type cmFDH contains no cystines, hence it is a good candidate to test the hypothesis that thermostability can be achieved by introducing new disulphide bridges. Three cysteine double mutants of cmFDH were designed, using a homology model reported previously, to introduce cystine bridges in the C-domain (T169C–T226C) in the N-domain (V88C–V112C) and between the two monomers (M156C–L159C) to form two cystine bridges across the dimer interface. These mutants were constructed and the proteins were over-expressed in E. coli. The mutants V88C–V112C and M156C–L159C lost FDH activity. The mutant T169C–T226C was both less active and less thermostable than wild-type FDH.     

Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.     

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity

To investigate the effect of natural pyrazinamidase (PncA) mutations on protein function, we analyzed expression and PncA activity of eight pncA point mutants identified in nineteen pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates. Among them, two mutants (Y99D and T135P) showed high expression level and solubility comparable to those of the wild-type PncA protein, two (K48E and G97D) displayed low expression level and solubility, and four (C14R, H51P, W68S, and A146V) were insoluble. Interestingly, when possible structural effects of these mutations were predicted by the CUPSAT program based on the proposed three-dimensional structure of M. tuberculosis PncA, only two highly soluble mutant proteins (Y99D and T135P) were predicted to be stabilizing and have favorable torsion angles. However, the others exhibiting either low solubility or precipitation were foreseen to be destabilizing and/or have unfavorable torsion angles, suggesting that the alterations could interfere with proper protein folding, thereby decreasing or depleting protein solubility. A PncA activity assay demonstrated that two mutants (G97D and T135P) showed virtually no activity, but two other mutants (K48E and Y99D) exhibited wild-type activity, indicating that the PncA residues (Cys14, His51, Trp68, Gly97, Thr135, and Ala146) may be important for PncA activity and/or proper protein folding.