Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease

Abstract

The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN, and d-DENN domains, is a GDP-GTP exchange factor (GEFs) for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation, and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2), nitrogen permease regulator 2 (NPR2), and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.

Keywords: ALS; C9ORF72; DENN domain; FTD; evolution; homology detection; longin domain; membrane trafficking.

Figure 1

Multiple sequence alignment and the core structure of Longin domains of the DENN module containing proteins. The secondary structure for the Longin domains is indicated above the alignment. The consensus in 75% of the sequences shown below is derived using the following amino acid classes: b, big (EFHIKLMQRWY); c, charged (DEHKR); h, hydrophobic (ACFGHILMTVWY); l, aliphatic (ILV); p, polar (CDEHKNQRST, on blue); s, small (ACDGNPSTV, on green). The numbers in bracket are indicative of the excluded residues from sequences. Long inserts of low-complexity sequences are highlighted in red on gray.

Figure 2

Multiple sequence alignment and the core structure of DENN domains of the DENN module containing proteins. The inferred secondary structure is indicated above the alignment. The consensus in 80% of the sequences shown below is derived using the following amino acid classes: b, big (EFHIKLMQRWY); c, charged (DEHKR); h, hydrophobic (ACFGHILMTVWY); l, aliphatic (ILV); p, polar (CDEHKNQRST, on blue); s, small (ACDGNPSTV, on green). The numbers in bracket are indicative of the excluded residues from sequences. Long inserts of low-complexity sequences are highlighted in red on gray.

Figure 3

Domain architectures of representatives of the DENN module containing proteins, including typical DENN proteins, and newly identified ones such as Folliculin (FLCN), SMCR8, C9ORF72, FNIP1/2, NPR2, and NPR3. For each family, both the human protein and an ortholog from a basal eukaryote are shown. Long inserts of low-complexity sequences within domains are shown as wavy lines. The sequences are indicated by protein names followed by species abbreviations and GenBank GIs.

Figure 4

Higher order relationships, domain architectures and cartoon representations of the DENN module. On the left is shown a tree depicting the inferred higher order relationships of the DENN modules. Lineages present in the last eukaryotic common ancestor are labeled “LECA.” Key events in the evolution of the module are labeled on the tree. Representative domain architectures of different DENN modules are shown in to the right of the figure. Cartoon representations of protein structures were derived from the PDB files labeled in the figure. These illustrate the common binding mode of the MglB/Longin/u-DENN domains with various GTPases (colored gray). They also depict the DENN and the d-DENN domains and their mode of interaction with GTPases.