Michael L. Skowyra

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infection in immunocompromised patients, which in severe cases leads to fatal meningoencephalitis. Cryptococcus exhibits unique glycobiology that plays important roles in pathogenesis. Unlike model yeast and other common fungal pathogens, Cryptococcus incorporates xylose, a five-carbon monosaccharide, into its glycans. One trimer motif, which consists of xylose in β-1,2 linkage to the reducing mannose of an α-1,3-mannose dimer, occurs in key cryptococcal glycoconjugates that include protein N- and O -linked glycans, glycosylinositol phosphorylceramides (GIPCs), and the capsule polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). We previously identified c ryptococcal β-1,2- x ylosyl t ransferase 1 (Cxt1), which catalyzes formation of this motif in GIPCs, GXM, and GXMGal. Here, we report the discovery of a second enzyme, c ryptococcal β-1,2- x ylosyl t ransferase 2 (Cxt2). Through characterization of cells that lack one or both corresponding genes ( CXT1 and CXT2 ), we have dissected the biological roles of these enzymes, which are overlapping but not identical. Notably, Cxt1 and Cxt2 co-localize in the Golgi, influence capsule in a strain-dependent manner, and together are responsible for all xylose addition to O -glycans. Overall, our work highlights unique roles of these two enzymes and fills a gap in understanding of cryptococcal glycan synthesis. IMPORTANCE: Cryptococcus neoformans is an opportunistic fungal pathogen that causes almost 150,000 deaths each year worldwide. Cryptococcus synthesizes unique glycan structures that play important roles in its biology and pathogenesis. One abundant component of these structures is xylose, a five-carbon monosaccharide. Because xylose is not used by many fungal organisms, including model yeast, we have limited information about how cells add it to their glycans. Here we report a xylosyltransferase enzyme that performs this function, and we characterize specific biological roles of this protein and a closely related one we discovered earlier. We find that these proteins together perform all detectable xylose addition to an important class of protein-linked glycans ( O -glycans). They also both participate in other synthetic processes, although this varies with the specific enzyme and strain background. These results contribute to our understanding of cryptococcal glycan synthesis and underscore the importance of testing multiple background strains.

Most proteins imported into peroxisomes use a carboxy-terminal PTS1 signal, which is recognized by soluble receptors that transport the cargo through a nuclear pore-like conduit in the peroxisomal membrane formed by the tyrosine and glycine-rich YG domain of PEX13. The receptors then return to the cytosol through a separate retrotranslocon. Some peroxisomal proteins instead use an amino-terminal PTS2 signal that is recognized by an adaptor called PEX7, but how they are imported is poorly understood. Here we show that PTS2 cargo is moved through the YG phase by PEX7 bound to a receptor. After cargo release inside peroxisomes, PEX7 returns to the cytosol by moving back on its own through the YG phase. The chaperone PEX39 then extracts PEX7 from the phase on the cytosolic side and helps to reload PEX7 with a new receptor and cargo to start another import cycle. Our results provide a comprehensive model of PTS2 protein import.

Nucleases activated by RNA are well known, and include eukaryotic RNA-silencing complexes and prokaryotic CRISPR-Cas systems. However, this and four other recent studies report the discovery of an RNA-activated protease {1-4}. In certain CRISPR-Cas systems called type III-E, a caspase-like protease has previously been found to associate with the CRISPR-Cas effector complex {5}. The CRISPR-Cas effector uses genomically encoded RNA to recognize and degrade complementary nucleic acids, and thereby confers immunity against viruses and other parasitic genetic elements. All five studies now show that binding of complementary RNA also activates the associated protease. Curiously, only imperfect complementarity activates the protease, perhaps to avoid activation by self RNA. The reason behind this conundrum is revealed by cryo-electron microscopy (cryo-EM) structures of the complex in different RNA-bound states. In the absence of complementary RNA, or when the complementarity is perfect, two key proteolytic residues are kept separated from each other. Imperfect complementarity displaces a regulatory helix, which then pulls the residues closer together. The consequences of activating the protease are unclear, but likely trigger additional immune responses. The studies show that the activated protease cleaves an endogenous bacterial protein, whose cleavage products may be cytotoxic and thus prevent infections from spreading to neighboring cells. This substrate protein also binds a transcription factor, which may be released upon cleavage to induce expression of alternative defense genes. Interestingly, the protease has recently been implicated in the proteolytic activation of bacterial gasdermins {6}, but whether this function is similarly triggered by foreign RNA remains to be explored. Activation of the protease by specific nucleic acids may have innovative biotechnological applications, for example in programmable RNA sensing.

Nucleases activated by RNA are well known, and include eukaryotic RNA-silencing complexes and prokaryotic CRISPR-Cas systems. However, this and four other recent studies report the discovery of an RNA-activated protease {1-4}. In certain CRISPR-Cas systems called type III-E, a caspase-like protease has previously been found to associate with the CRISPR-Cas effector complex {5}. The CRISPR-Cas effector uses genomically encoded RNA to recognize and degrade complementary nucleic acids, and thereby confers immunity against viruses and other parasitic genetic elements. All five studies now show that binding of complementary RNA also activates the associated protease. Curiously, only imperfect complementarity activates the protease, perhaps to avoid activation by self RNA. The reason behind this conundrum is revealed by cryo-electron microscopy (cryo-EM) structures of the complex in different RNA-bound states. In the absence of complementary RNA, or when the complementarity is perfect, two key proteolytic residues are kept separated from each other. Imperfect complementarity displaces a regulatory helix, which then pulls the residues closer together. The consequences of activating the protease are unclear, but likely trigger additional immune responses. The studies show that the activated protease cleaves an endogenous bacterial protein, whose cleavage products may be cytotoxic and thus prevent infections from spreading to neighboring cells. This substrate protein also binds a transcription factor, which may be released upon cleavage to induce expression of alternative defense genes. Interestingly, the protease has recently been implicated in the proteolytic activation of bacterial gasdermins {6}, but whether this function is similarly triggered by foreign RNA remains to be explored. Activation of the protease by specific nucleic acids may have innovative biotechnological applications, for example in programmable RNA sensing.

Nucleases activated by RNA are well known, and include eukaryotic RNA-silencing complexes and prokaryotic CRISPR-Cas systems. However, this and four other recent studies report the discovery of an RNA-activated protease {1-4}. In certain CRISPR-Cas systems called type III-E, a caspase-like protease has previously been found to associate with the CRISPR-Cas effector complex {5}. The CRISPR-Cas effector uses genomically encoded RNA to recognize and degrade complementary nucleic acids, and thereby confers immunity against viruses and other parasitic genetic elements. All five studies now show that binding of complementary RNA also activates the associated protease. Curiously, only imperfect complementarity activates the protease, perhaps to avoid activation by self RNA. The reason behind this conundrum is revealed by cryo-electron microscopy (cryo-EM) structures of the complex in different RNA-bound states. In the absence of complementary RNA, or when the complementarity is perfect, two key proteolytic residues are kept separated from each other. Imperfect complementarity displaces a regulatory helix, which then pulls the residues closer together. The consequences of activating the protease are unclear, but likely trigger additional immune responses. The studies show that the activated protease cleaves an endogenous bacterial protein, whose cleavage products may be cytotoxic and thus prevent infections from spreading to neighboring cells. This substrate protein also binds a transcription factor, which may be released upon cleavage to induce expression of alternative defense genes. Interestingly, the protease has recently been implicated in the proteolytic activation of bacterial gasdermins {6}, but whether this function is similarly triggered by foreign RNA remains to be explored. Activation of the protease by specific nucleic acids may have innovative biotechnological applications, for example in programmable RNA sensing.

Nucleases activated by RNA are well known, and include eukaryotic RNA-silencing complexes and prokaryotic CRISPR-Cas systems. However, this and four other recent studies report the discovery of an RNA-activated protease {1-4}. In certain CRISPR-Cas systems called type III-E, a caspase-like protease has previously been found to associate with the CRISPR-Cas effector complex {5}. The CRISPR-Cas effector uses genomically encoded RNA to recognize and degrade complementary nucleic acids, and thereby confers immunity against viruses and other parasitic genetic elements. All five studies now show that binding of complementary RNA also activates the associated protease. Curiously, only imperfect complementarity activates the protease, perhaps to avoid activation by self RNA. The reason behind this conundrum is revealed by cryo-electron microscopy (cryo-EM) structures of the complex in different RNA-bound states. In the absence of complementary RNA, or when the complementarity is perfect, two key proteolytic residues are kept separated from each other. Imperfect complementarity displaces a regulatory helix, which then pulls the residues closer together. The consequences of activating the protease are unclear, but likely trigger additional immune responses. The studies show that the activated protease cleaves an endogenous bacterial protein, whose cleavage products may be cytotoxic and thus prevent infections from spreading to neighboring cells. This substrate protein also binds a transcription factor, which may be released upon cleavage to induce expression of alternative defense genes. Interestingly, the protease has recently been implicated in the proteolytic activation of bacterial gasdermins {6}, but whether this function is similarly triggered by foreign RNA remains to be explored. Activation of the protease by specific nucleic acids may have innovative biotechnological applications, for example in programmable RNA sensing.

Nucleases activated by RNA are well known, and include eukaryotic RNA-silencing complexes and prokaryotic CRISPR-Cas systems. However, this and four other recent studies report the discovery of an RNA-activated protease {1-4}. In certain CRISPR-Cas systems called type III-E, a caspase-like protease has previously been found to associate with the CRISPR-Cas effector complex {5}. The CRISPR-Cas effector uses genomically encoded RNA to recognize and degrade complementary nucleic acids, and thereby confers immunity against viruses and other parasitic genetic elements. All five studies now show that binding of complementary RNA also activates the associated protease. Curiously, only imperfect complementarity activates the protease, perhaps to avoid activation by self RNA. The reason behind this conundrum is revealed by cryo-electron microscopy (cryo-EM) structures of the complex in different RNA-bound states. In the absence of complementary RNA, or when the complementarity is perfect, two key proteolytic residues are kept separated from each other. Imperfect complementarity displaces a regulatory helix, which then pulls the residues closer together. The consequences of activating the protease are unclear, but likely trigger additional immune responses. The studies show that the activated protease cleaves an endogenous bacterial protein, whose cleavage products may be cytotoxic and thus prevent infections from spreading to neighboring cells. This substrate protein also binds a transcription factor, which may be released upon cleavage to induce expression of alternative defense genes. Interestingly, the protease has recently been implicated in the proteolytic activation of bacterial gasdermins {6}, but whether this function is similarly triggered by foreign RNA remains to be explored. Activation of the protease by specific nucleic acids may have innovative biotechnological applications, for example in programmable RNA sensing.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)– and glycine (G)–rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.

In many animals, proteins are targeted to lysosomes by the attachment of mannose-6-phosphate (M6P) in the Golgi. The modification occurs by the consecutive action of two enzymes, GlcNAc-1-phosphotransferase (GNPT) and M6P-uncovering enzyme (UCE), but the regulation of these enzymes is poorly understood. This paper reports the discovery of a novel factor called LYSET that is required for M6P installation in mammals. LYSET emerged from an elegant CRISPR screen looking for genes that allow extracellular proteins to be broken down in lysosomes as a source of amino acids. Loss of LYSET caused a near-complete block of lysosomal degradation, which mirrored the phenotype caused by loss of GNPT, the first enzyme that installs M6P in the Golgi. Indeed, M6P was undetectable in cells lacking LYSET and hydrolytic enzymes failed to reach lysosomes. By an impressive series of experiments, the authors found that LYSET retains GNPT in the Golgi by preventing the enzyme from being degraded in lysosomes. Without LYSET, a hydrophilic transmembrane segment in GNPT causes the enzyme to be mistargeted to lysosomes. Being a small membrane protein, LYSET might stabilize this transmembrane segment of GNPT, but how this would occur is unclear. The two proteins associate with each other by immunoprecipitation, but a direct interaction was not demonstrated. Interestingly, the authors note that in certain animals which lack LYSET, GNPT also has a more hydrophobic transmembrane segment. While this observation supports the proposed model, it raises the question of why LYSET is dispensable in some animals but required in others. Perhaps, LYSET is needed to regulate lysosomal protein trafficking by controlling the abundance of GNPT, but this hypothesis remains to be tested. Nevertheless, defects in lysosomal protein trafficking likely underlie the skeletal dysplasias that result from mutations in LYSET (formerly known as TMEM251). The study also suggests that inhibiting or downregulating LYSET might help treat certain cancers that rely disproportionately on lysosomal degradation for their survival.